Diketopiperazine Forming Dipeptidyl Linker

ABSTRACT

The invention relates to a method for homogeneous solution phase peptide synthesis (HSPPS) of a N-terminal peptide fragment PEP-N and a C-terminal peptide fragment C-PEP, with C-PEP carrying a specific diketopiperazine (DKP) comprising C-terminal protecting group, which contains a handle group HG, with HG being connected to the C-terminus of the peptide fragmcnt; thereby this specific DKP comprising C-terminal protecting group can be selectively cleaved from the peptide as a conventionally used C-terminal protecting group. By the use of this DKP and HG comprising C-terminal protecting group, certain process steps in convergent peptide synthesis based on a combination of HSPPS and solid phase peptide synthesis (SPPS) can be avoided. The invention relates further to a method for the preparation of such specifically protected fragment C-PEP by SPPS by using a linker comprising a specific dipeptide and HG for connecting the growing peptide chain to the resin, which linker forms said DKP group, when the peptide fragment C-PEP is cleaved from the supporting resin; and further to the intermediates of the preparation method.

RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 13/881,883, filed on Apr. 26, 2013, and claimspriority to PCT International Application No. PCT/EP2011/005280, havinga filing date of Oct. 20, 2011, which claims filing benefit of EuropeanPatent Application EP10014114.2 having a filing date of Oct. 29, 2010;EP10015436.8 and EP10015434.3 both having a filing date of Dec. 8, 2010;EPI 1001442.0 having a filing date of Feb. 22, 2011; EP 1004819.6 havinga filing date of Jun. 14, 2011 and U.S. Provisional Application Ser. No.61/496,655 having a filing date of Jun. 14, 2011, which are incorporatedherein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 13, 2013, isnamed LZA-8-PCT-US_SL.txt and is 2,937 bytes in size.

The invention relates to a method for homogeneous solution phase peptidesynthesis (HSPPS) of a N-terminal peptide fragment PEP-N and aC-terminal peptide fragment C-PEP, with C-PEP carrying a specificdiketopiperazine (DKP) comprising C-terminal protecting group, whichcontains a handle group HG, with HG being connected to the C-terminus ofthe peptide fragment; thereby this specific DKP comprising C-terminalprotecting group can be selectively cleaved from the peptide as aconventionally used C-terminal protecting group. By the use of this DKPand HG comprising C-terminal protecting group, certain process steps inconvergent peptide synthesis based on a combination of HSPPS and solidphase peptide synthesis (SPPS) can be avoided.

The invention relates further to a method for the preparation of suchspecifically protected fragment C-PEP by SPPS by using a linkercomprising a specific dipeptide and HG for connecting the growingpeptide chain to the resin, which linker forms said DKP group, when thepeptide fragment C-PEP is cleaved from the supporting resin; and furtherto the intermediates of the preparation method.

In this text, the nomenclature of amino acids and of peptides is usedaccording to “Nomenclature and symbolism for amino acids and peptides”,Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984, if not otherwisestated.

The following abbreviations have the meaning as given in the followinglist, if not otherwise stated:

CTC chlorotrityl chlorideAlloc allyloxycarbonylBoc tert-butoxycarbonylBsmoc 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonylBzl or Bn benzylcHx cyclohexylCt C terminalDpr 2,3-diaminopropanoic acidDde N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethylivDde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methylbutylDdz alpha,alpha-dimethyl-3,5-dimethoxybenzyloxycarbonylDKP 2,5-diketopiperazineDmab dimethylaminoborane

Fm 9-Fluorenylmethyl

Fmoc N-(fluorenyl-9-methoxycarbonyl)Hpr piperidine-2-carboxylic acid, homoprolineHSHSPPS hybrid solid and homogenous solution phase peptide synthesisHSPPS homogenous solution phase peptide synthesisHyp trans-4-hydroxyprolineMmt 4-methoxytritylMpe 3-methylpent-3-yl,Mtt 4-methyltritylOrn ornithinePbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonylPG protecting group2-PhiPr 2-phenylisopropylPmc 2,2,5,7,8-penta-methylchroman-6-sulfonylpNO₂Z nitrobenzyloxycarbonyl

Py Pyridine

SPPS solid phase peptide synthesistBu tert-butyl

TES SiEt3, Triethylsilyl

TFA Trifluoroacetic acidTfac trifluoroacetylTrt or Tr triphenylmethyl or tritylZ benzyloxycarbonyl

The terms “fragment” and “peptide fragment” are used synonymously, ifnot otherwise stated. The terms “handle” and “handle group”, e.g.“Fmoc-Rink amide handle group”, “Rink amide handle” or “Rink amidehandle group”, and the term “linker”, e.g. “Fmoc-Rink amide linker” orFmoc-Rink-OH, are often used synonymously, if not stated otherwise.

Peptides are often prepared by hybrid solid and homogenous solutionphase peptide synthesis IISHSPPS: firstly two or more peptide fragmentsare prepared by solid phase peptide synthesis SPPS, which are thereaftercoupled in solution phase by homogenous solution phase peptide synthesisHSPPS to provide for the desired target peptide.

This approach is particularly attractive for the commercial scalepreparation of large peptides as it combines the advantages of both theSPPS and the HSPPS. In particular, the SPPS of fragments can bedeveloped and scaled-up rapidly and avoids many of the solubilityproblems often encountered in HSPPS of relatively long fragments.Production cycle times are short compared to solution phasemethodologies. In addition, yields and purities are often higher becauseof the use of excess reagents, especially during the coupling reactions,which often results in intermediates that do not require purification.After optimization of selection of the sequences of the fragments madeby SPPS, the final stages of the process can be scaled-up byconventional HSPPS methodologies. These final stages of the process arethe fragment coupling and the final deprotection of the amino acidresidues, i.e. the deprotection of the side chains and of the N- andC-terminus, both being performed in solution. Thus, when applying theHSHSPPS synthesis, the advantages of the SPPS, i.e. rapid synthesis offragments with high purities, and the advantages of solution-phasesynthesis, i.e. full monitoring of coupling reactions and isolation andoptional purification including full characterization of the formedintermediate fragments, can be exploited in order to efficiently producepeptides, especially on commercial scale.

In HSHSPPS, always at least two fragments PEP-N and C-PEP prepared bySPPS are coupled in solution phase to provide the desired peptide PEP,which is either the final peptide or again an intermediate peptidefragment, which again thereafter is coupled with a third peptidefragment, and so on. Fragment PEP-N presents herein the N-terminus ofpeptide PEP, fragment C-PEP presents the C-terminus of peptide PEP, andtherefore the C-terminus of fragment PEP-N is coupled with theN-terminus of fragment C-PEP to provide peptide PEP. It is necessary,that the N-terminus of fragment PEP-N is protected as well as theC-terminus of fragment C-PEP is protected during solution phase couplingin order to avoid undesired coupling of fragment PEP-N with fragmentPEP-N, of fragment C-PEP with fragment C-PEP, or of fragment C-PEP withfragment PEP-N in the wrong direction. This N-terminally protectedpeptide fragment PEP-N is in the following also called PEP-N, if nototherwise stated. The fragment C-PEP, prepared by SPPS on a supportingresin, will carry an N-terminal protecting group after the addition ofthe last amino acid residue, and will then be cleaved from thesupporting resin in a final step. This cleavage results usually in afragment C-PEP with an unprotected C-terminus, which must be protectedin a separate step, before fragment C-PEP can be coupled in HSPPS withfragment PEP-N. Actually, this necessary protection of the C-terminus offragment C-PEP comprises not only one step, but several steps such asreaction, purification and isolation, possibly with another subsequentpurification and isolation.

In case the target peptide PEP to be prepared is a peptide amidePEP-NH₂, i.e. with the C-terminus being a carboxamide group, theC-terminus of the respective fragment C-PEP-NH₂normally does not need tobe protected during fragment coupling in HSPPS, since the carboxamidegroup itself acts as a protecting group. While a fragment C-PEP-OH, withthe C-terminus being the carboxylic acid, can be easily obtained afterSPPS by use of a resin which forms the carboxylic acid group aftercleavage, the use of a resin which forms the carboxamide group aftercleavage, e.g. the Sieber amide resin, causes problems due to partialside chain deprotection of the fragment C-PEP-NH₂ during cleavage, sincecleavage from amide resins typically requires acidic conditions, such asthe use of 3 to 5% by weight of TFA in a solvent, and side chainprotecting groups, such as Trt in case of Fmoc/Trt SPPS (for exampleHis(Trt)) or such as acetale in pseudo-proline derivatives (i.e.Fmoc-Ser(tBu)-Thr(psi^(Mc),^(Me)pro)-OH), are not completely stableunder such cleavage conditions, which results in partial loss of theside chain protecting groups. Therefore for preparing fragmentC-PEP-NH₂, it is common to start the SPPS with the amino acid second inposition from the C-terminal amino acid residue of the desired fragmentC-PEP-NH₂ and not with the C-terminal amino acid itself of fragmentC-PEP-NH₂, and with a resin which affords a carboxylic acid asC-terminus after cleavage. Cleavage from the resin affords therefore afragment C—OH without the C-terminal amino acid of the desired fragmentC-PEP-NH₂, and with the C-terminus of this fragment C—OH being the aminoacid of the second position from the C-terminus of the finally desiredfragment C-PEP-NH₂ and bearing a carboxylic acid group. The missingC-terminal amino acid of fragment C-PEP-NH₂ is then separately coupledto the fragment C—OH in form of its amide H-Xaa-NH₂ in solution phase.

WO 90/09395 discloses the use of a cleavable linker between peptide andthe supporting resin, which forms a diketopiperazine (DKP) linker groupwhen cleaved from the resin, wherein the DKP group is connected to thepeptide via an amide bond between the epsilon amino group of a Lys inthe linker group and the C-terminus of the peptide. This DKP linkergroup cannot be removed selectively from the peptide at a later stage.Thus, it does not allow for the preparation of fully protectedC-terminal fragments with unprotected N-terminus suitable in HSPPS.Furthermore, the linker group of WO 90/09395 does not allow for thepreparation of natural or unmodified peptides. It is only suitable forthe synthesis of permanently C-terminally modified peptides, since anypeptide cleaved from the resin always carries a DKP linker group at itsC-terminus, which is not cleavable without cleaving the other peptidebonds of the peptide. Another disadvantage is the restriction of itscleavage to the use oftrifluoroacetic acid (TFA) during the cleavagestep, which implies the partial or total removal of any tBu, Boc, Trt orAcetale based protecting groups of the side chains of the amino acidresidues of the peptide, thereby restricting its use to the preparationof either peptides with unprotected side chains or to side chainprotecting groups other than tBu, Boc, Trt and Acetale.

There was a need to simplify the procedure of HSHSPPS by reducing thenumber of steps in the reaction sequence.

Surprisingly, this can be achieved by using a specific diketopiperazinegroup forming dipeptidyl linker in the SPPS used to prepare the fragmentC-PEP, which carries a specific diketopiperazine comprising C-terminalprotecting group, together with an appropriate combination of thedifferent types of protecting groups and a specific chemical nature ofthe connection of the linker to the fragment C-PEP providing specificcleavage possibility of the linker from the fragment.

Protecting groups (PG), be it for protecting functional groups in sidechains of amino acid or for the protection of N-terminal amino groups orC-terminal carboxy groups of amino acids or peptides, are for thepurpose of this invention classified into four different groups:

1. basic cleavable type protecting groups, in the following called“basic type PGs”,2. strong acid cleavable type protecting groups, in the following called“strong type PGs”,3. weak acid cleavable type protecting groups, in the following called“weak type PGs”, and4. reductively cleavable type protecting groups. in the following called“reductive type PGs”, with the two groups “strong type PGs” and “weaktype PGs” also collectively called “acid cleavable type protectinggroups” or “acid type PGs”.

Within the meaning of this invention, any PG is classified by thefollowing four classification reaction conditions. The classification isdone using a CTC resin with a loading capacity of 1.5 to 1.7 mmol per gresin, the resin being loaded with only one amino acid carrying therespective PG which is to be classified. The term “part” in thefollowing four classification procedures is meant to be a factor of theparts by weight of the loaded CTC resin starting material, if nototherwise stated.

1. Classification reaction conditions for basic type PG, in thefollowing text called “basic classification conditions”:

Treatment for 25+/5 min at 25+/−5° C. of the resin loaded with the basictype PG carrying amino acid with 7+/−1 parts of a cleaving solution, thecleaving solution consisting of 22.5 +/−2.5% by weight solution ofpiperidine in dimethylformamide (DMF), the % by weight being based onthe total weight of the cleaving solution.

2. Classification reaction conditions for strong type PGs, in thefollowing text called “strong classification conditions”:Treatment for 25+/5 min at 25+/−5° C. of the resin loaded with thestrong type PG carrying amino acid with 7+/−1 parts of a cleavingsolution, the cleaving solution consisting of 85+/−5% by weight solutionof trifluoro acetic acid (TFA) in dichloromethane (DCM), the % by weightbeing based on the total weight of the cleaving solution.3. Classification reaction conditions for weak type PGs, in thefollowing text called “weak classification conditions”:Treatment for 25+/5 min at 25+/−5° C. of the resin loaded with the weaktype PG carrying amino acid with 7+/−1 parts of a cleaving solution, thecleaving solution consisting of 2+/−1 5% by weight solution of TFA inDCM, the % by weight being based on the total weight of the cleavingsolution.4. Classification reaction conditions for reductive type PGs, in thefollowing text called “reductive classification conditions”:Treatment for 30+/5 min at 25+/−5° C. of the resin loaded with thereductive type PG carrying amino acid with 7+/−1 parts of DMF, with 0.1mol equivalent of a soluble organic Pd(0) catalyst, preferablyPd[PPh_(3]4), dissolved in the DMF, the mol equivalent being based onthe mol of cleavable groups loaded on the resin.

PGs and typical reaction conditions and parameters and reagents forcleaving PGs, which are conventionally used in peptide chemistry, areknown in the art, e.g. T. W. Greene, P. G. M. Wuts “Protective Groups inOrganic Synthesis” John Wiley & Sons, Inc., 1999; or Lloyd-Williams, P.,Albericio, F., Giralt, E., “Chemical Approaches to the Synthesis ofPeptides and Proteins” CRC: Boca Raton, Fla., 1997.

Basic type PGs are preferably cleaved under following possible reactionconditions, in the following text called “basic cleaving conditions”:

Basic cleaving conditions involve treatment of the respective materialwith a basic cleaving solution. The basic cleaving solution comprises abasic reagent and a solvent. Preferably, the basic cleaving solutionconsists of a basic reagent and a solvent. If the basic reagent isliquid at the temperature, at which the basic cleaving is done, thebasic reagent can also act simultaneously as the solvent, i.e. nosolvent different from the basic reagent is used.

Basic reagents are preferably secondary amines, more preferably thebasic reagent is selected from the group consisting of piperidine,4-(aminomethyl)piperidine, tris(2-aminoethyl)amine, morpholine,dicyclohexylamine, 1,3-cyclohexanebis(methylamine)piperazine,1,8-diazabicyclo[5.4.0]undec-7-ene and mixtures thereof. Even morepreferably, the basic reagent is piperidine.

The basic cleaving solution can also comprise an additive, the additivepreferably selected from the group consisting of6-chloro-1-hydroxy-benzotriazole, 2,4-dinitrophenol, picric acid,1-hydroxy-7-azabenzotriazole, 1-hydroxy-benzotriazole and ethyl2-cyano-2-hydroxyimino-acetate and mixtures thereof.

Preferably, the solvent is selected from the group consisting ofdimethylsulfoxide (DMSO), dioxane, tetrahydrofuran (THF),1-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF),N,N-dimethylacetamide (DMA), pyridine, dichloromethane (DCM),dichloroethane (DCE), chloroform, dioxane, tetrahydropyran, ethylacetate, toluene, acetonitrile and mixtures thereof; more preferably thesolvent is 1-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF) ora mixture thereof.

The term “part” in this description of basic cleaving conditions ismeant to be a factor of the parts by weight of the treated materialcarrying the basic type PG(s).

Preferably, of from 5 to 20 parts, more preferably of from 5 to 15 partsof basic cleaving solution are used.

Preferably, the amount of basic reagent is of from 1 to 30% by weight,more preferably of from 10 to 25% by weight, even more preferably offrom 15 to 20% by weight, with the % by weight being based on the totalweight of the basic cleaving solution.

Preferably, basic cleaving is done at a temperature of from 10 to 50°C., more preferably of from 10 to 30° C., even more preferably of from15 to 25° C.

Preferably, basic cleaving is done at atmospheric pressure.

Preferably, the reaction time for basic cleaving is of from 5 min to 2h, more preferably of from 10 min to 1 h, even more preferably of from15 min to 30 min.

Strong type PGs are preferably cleaved under the following possiblereaction conditions, in the following text called “strong cleavingconditions”:

Strong cleaving conditions involve treatment of the respective materialwith a strong cleaving solution. The strong cleaving solution comprisesan acidolytic reagent. Acidolytic reagents are preferably selected fromthe group consisting of hydrogen acids, such as trifluoroacetic acid(TFA), hydrochloric acid (HCl), aqueous hydrochloric acid (HCl), liquidhydrofluoric acid (HF) or trifluoromethanesulfonic acid, Lewis acids,such as trifluoroborate diethyl ether adduct or trimethylsilylbromid,and mixtures thereof.

The strong cleaving solution preferably comprises one or morescavengers, the scavengers being selected from the group consisting ofdithiothreitol (DTT), ethanedithiol (EDT), dimethylsulfide (DMS),triisopropylsilane (TIS), triethylsilane (TES), 1,3-dimethoxybenzene(DMB), phenol, anisole, p-cresol and mixtures thereof.

The strong cleaving solution can also comprise water, a solvent or amixture thereof, the solvent being stable under strong cleavingconditions.

Preferably, solvents are selected from the group consisting ofdichloromethane, dichloroethane, acetonitrile, toluene,tetrahydrofurane, TFA, dioxane and mixtures thereof.

More preferably, the acidolytic reagent acts simultaneously as solvent,so no further solvent is needed.

The term “part” in this description of strong cleaving solution is meantto be a factor of the parts by weight of the treated material carryingthe strong type PG(s).

Preferably, of from 10 to 30 parts, more preferably of from 15 to 25parts, even more preferably of from 19 to 21 parts of strong cleavingsolution are used.

Preferably, the amount of acidolytic reagent is of from 30 to 100% byweight, more preferably of from 50 to 100% by weight, even morepreferably of from 70 to 100% by weight, especially of from 80 to 100%by weight, with the % by weight being based on the total weight of thestrong cleaving solution.

Preferably, of from 1 to 25% by weight of total amount of scavenger isused, more preferably of from 5 to 15% by weight, with the % by weightbased on the total weight of the strong cleaving solution.

Preferably, strong cleaving is done at a temperature of from −10 to 30°C., more preferably of from −10 to 30° C., even more preferably of from5 to 15° C.

Preferably, strong cleaving is done at atmospheric pressure.

Preferably, the reaction time for strong cleaving is of from 30 min to20 h, more preferably of from 1 h to 10 h, even more preferably of from1 h to 5 h.

Weak type PGs are preferably cleaved under the following possiblereaction conditions, in the following text called “weak cleavingconditions”:

Weak cleaving conditions involve treatment of the respective materialwith a weak cleaving solution. The weak cleaving solution comprises anacidolytic reagent. The acidolytic reagent is preferably selected fromthe group consisting of hydrogen acids, such as trifluoroacetic acid(TFA), trifluoroethanol (TFE), hydrochloric acid (HCl), acetic acid(AcOH), mixtures thereof and/or with water.

The weak cleaving solution also comprises water, a solvent or a mixturethereof, the solvent being stable under weak cleaving conditions.

Preferably, solvents are selected from the group consisting ofdichloromethane, dichloroethane, acetonitrile, toluene,tetrahydrofurane, TFA, dioxane and mixtures thereof.

The term “part” in this description of weak cleaving solution is meantto be a factor of the parts by weight of the treated material carryingthe weak type PG(s).

Preferably, of from 4 to 20 parts, more preferably of from 5 to 10parts, of weak cleaving solution are used.

Preferably, the amount of acidolytic reagent is of from 0.01 to 5% byweight, more preferably of from 0.1 to 5% by weight, even morepreferably of from 0.15 to 3% by weight, with the % by weight beingbased on the total weight of the weak cleaving solution.

Preferably, weak cleaving is done at a temperature of from 10 to 50° C.,more preferably of from 20 to 40° C., even more preferably of from 25 to35° C.

Preferably, weak cleaving is done at atmospheric pressure.

Preferably, the reaction time for weak cleaving is of from 5 min to 2 h,more preferably of from 10 min to 1 h, even more preferably of from 10min to 30 min.

The weak type PG can be subclassified into further groups, these groupsbeing differentiated from one another and can be aligned consecutivelyaccording to the amount of acid necessary for cleavage. According toabove definition of weak classification conditions, all weak type PGscan be cleaved using 2+/−1% by weight solution of TFA in DCM, the % byweight being based on the total weight of the cleaving solution. A weaktype PG, which is only cleaved by a solution of at least 1% by weight ofTFA in DCM, but not by a solution with less amount of TFA, is called“weak 1 type PG” and the cleaving conditions are called “weak 1 cleavingconditions”;

a weak type PG, which is cleaved already by a solution of at least 0.1%by weight of TFA in DCM, but not by a solution with less amount of TFA,is called “weak 2 type PG” and the cleaving conditions are called “weak2 conditions”;a weak type PG, which is cleaved already by a solution of at least 0.01%by weight of TFA in DCM, is called “weak 3 type PG” and the cleavingconditions are called “weak 3 conditions”; the % by weight being basedon the total weight of the cleaving solution.

Reductive type PGs are preferably cleaved under the following possiblereaction conditions, in the following text called “reductive cleavingconditions”:

Reductive cleaving conditions involve treatment of the respectivematerial with a reductive cleaving solution. The reductive cleavingsolution comprises a catalyst, an additive and a solvent.

The catalysts are preferably selected from the group consisting oforganic derivatives of Pd(0) and organic derivates of Pd(II),

more preferably selected from the group consisting of Pd[PPh₃]₄,PdCl₂[PPh₃]₂, Pd[OAc]₂[P(2,4-xyloyl)₃]₂, Pd[OAc]₂[P(ortho-tolyl)₃]₂,

-   in situ prepared Pd(0) catalysts, prepared by mixing less stably    coordinated Pd-complexes with ligands, such as PdCl₂(PPh₃)2/PPh₃,    PdCl₂(PPh₃)2/P(ortho-tolyl)₃, Pd(DBA)₂/P(ortho-tolyl)₃ or    Pd[P(ortho-tolyl)₃]₂, Pd(OAc)/triethyl-phosphite, Pd(OAc)₂/PPh₃or    Pd(OAc)₂/P(ortho-tolyl)₃,    and mixtures thereof;    even more preferably selected from the group consisting of    Pd[PPh_(3]4), PdCl₂[PPh_(3]2), Pd[OAc]₂[P(2,4-xyloyl)_(3]2),    Pd[OAc]₂[P(ortho-tolyl)_(3]2) and mixtures thereof.

The additive is preferably selected from the group consisting ofdimethylbarbituric acid, thiosalicylic acid, N-methylaniline, Bu₄N⁺BH₄⁻, NH₃BH₃, Me₂NHBH₃, tBu-NH₂BH₃, Me₃NBH₃, PyBH₃, HCOOH/DIEA,diethydithiocarbamate sodium, dimedone, morpholine, AcOH/NMM,phenylsilane, sulfinic acids comprising PhSO₂H, tolSO₂Na, sodium2-ethylhexanoate (SEH), sodium 2-thiophenesulfinate (STS), sodium4-chloro-3-nitrobenzenesulfinate (SCNBS) and i-BuSO₂Na, and mixturesthereof; more preferably the additive is tolSO₂Na.

Preferably, the solvent is selected from the group consisting ofdimethylsulfoxide (DMSO), dioxane, tetrahydrofuran (THF),1-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF),N,N-dimethylacetamide (DMA), pyridine, dichloromethane (DCM),dichloroethane (DCE), chloroform, dioxane, tetrahydropyran, ethylacetate, toluene, acetonitrile and mixtures thereof; more preferably thesolvent is 1-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF) ora mixture thereof.

Preferably, the catalyst is dissolvable in the solvent and is dissolvedin the solvent.

The term “part” in this description of reductive cleaving conditions ismeant to be a factor of the parts by weight of the treated materialcarrying the reductive type PG(s).

Preferably, of from 4 to 20 parts, more preferably of from 5 to 10parts, of reductive cleaving solution are used.

Preferably, 0.001 to 1 mol equivalents, more preferably 0.01 to 0.05 molequivalents, of catalyst are used, the mol equivalent being based on themol of reductively cleavable groups loaded on the resin.

Preferably, 1 to 10 mol equivalents, more preferably 1.5 to 5 molequivalents, of additive are used, the mol equivalent being based on themol of reductively cleavable groups loaded on the resin.

Preferably, reductive cleaving is done at a temperature of from 10 to60° C., more preferably of from 30 to 50° C., even more preferably offrom 35 to 45° C.

Preferably, reductive cleaving is done at atmospheric pressure.

Preferably, the reaction time for reductive cleaving is of from 15 minto 10 h, more preferably of from 30 min to 4 h, even more preferably offrom 30 min to 2 h.

Preferably, the reductive cleaving solution has to be protected form thelight. Preferably, reductive cleaving is done in a container made ofmetal.

-   The basic type PGs are not cleavable by strong or weak cleaving    conditions.-   Preferably, the basic type PGs are not cleavable by strong, weak or    reductive cleaving conditions.-   The strong type PGs are not cleavable by weak or basic cleaving    conditions.-   Preferably, the strong type PGs are not cleavable by weak, basic or    reductive cleaving conditions.-   The weak type PGs are not cleavable by basic cleaving conditions,    but they are cleavable by strong cleaving conditions.-   Preferably, the weak type PGs are not cleavable by basic or    reductive cleaving conditions, but they are cleavable by strong    cleaving conditions.-   The weak 1 type PGs are not cleavable by weak 2 or weak 3 cleaving    conditions;-   the weak 2 type PGs are cleavable by weak 1 cleaving conditions, but    not by weak 3 cleaving conditions;-   the weak 3 type PG are cleavable by weak 1 and by weak 2 cleaving    conditions.-   preferably, the weak 1, 2 and 3 type PGs are also not cleavable by    basic or reductive cleaving conditions.-   Preferably, reductive type PGs are not cleavable by strong, weak and    basic cleaving conditions, these are called “exclusively reductive    type PGs”.-   Reductive type PGs, which are not cleavable by weak and basic    cleaving conditions, but which are cleavable by strong cleaving    conditions; these PGs are called “mixed type PGs”.

The connection of the linker to the peptide can also be classified to becleavable under one of these four cleaving conditions.

The connection of an amino acid to a resin can also be classified to becleavable under one of these four cleaving conditions.

-   Preferably, a basic type PG is selected from the group consisting of    Fmoc, Bsmoc, Tfac, Dde, Dmab and cHx.-   Preferably, a strong type PG is selected from the group consisting    of Boc, tBu, Pmc, Mpe, Pbf, Z, Bzl, cHx, pNO₂Z and Ddz.-   Preferably, a weak type PG is selected from the group consisting of    Trt, Mmt, Mtt, acetale and 2-PhiPr.-   If a weak type PG is actually a weak 1 type PG or a weak 2 type PG,    depends on the side chain group which it protects.-   Preferably, a reductive type PG is selected from the group    consisting of Alloc, Allyl, ivDde and Z.    More preferably, a basic type PG is Fmoc.    More preferably, a strong type PG is Boc.    More preferably, a weak type PG is Trt.    More preferably, a reductive type PG is Alloc.

In conventional SPPS, the peptide is cleaved from the resin after theSPPS is finished, the cleavage resulting in a peptide with a C-terminusin form of a free carboxylic acid group or in form of a carboxamide,depending on the resin and a possible handle used in SPPS. If thispeptide with a free carboxylic group at its C-terminus is to be used inHSPPS as the C-terminal peptide fragment C-PEP, this free carboxylicacid group has firstly to be protected, before the peptide can be usedin HSPPS. This protection of the free C-terminus needs several processsteps (reaction, isolation, perhaps purification).

The instant invention discloses a method for reducing these stepsnecessary for protecting a free carboxylic acid at the C-terminus of thefragment resulting from cleavage of the fragment from the resin afterSPPS. This is achieved by using said diketopiperazine forming dipeptidyllinker to couple in the SPPS the first amino acid XaaC⁽¹⁾ of the desiredfragment C-PEP via said linker onto the resin support, which linkerforms a diketopiperazine residue comprising C-terminal protecting group,when the SPPS is finished and the synthesized fragment C-PEP is beingcleaved from the resin. The diketopiperazine forming dipeptidyl linkercomprises a dipeptide moiety, whose first amino acid Xaa1 is via itscarboxylic acid group connected to the resin, and whose second aminoacid Xaa2 is via its side chain connected a handle group HG, whichhandle group HG is connected to the peptidyl radical, and Xaa2 carriesan N-terminal protecting group PG2.

The formation of said diketopiperazine residue comprising C-terminalprotecting group is achieved by cleaving the protecting group PG2 ofXaa2, thereby making an intramolecular ring closure between Xaa2 andXaa1 possible, which ring closure forms said diketopiperazine residueand simultaneously cleaves Xaa1 from the resin.

This diketopiperazine residue comprising C-terminal protecting group,formed by the cleavage from the resin, remains connected to theC-terminus of the fragment C-PEP after cleavage from the resin and actsthereby as a protecting group of the C-terminus of the fragment C-PEP,which can therefore directly be used in HSPPS. After coupling of thisC-terminal fragment C-PEP with an N-terminal peptide fragment PEP-N byHSPPS to yield the peptide PEP, the diketopiperazine residue comprisingC-terminal protecting group is cleaved from the peptide PEP, preferablysimultaneously with the deprotection of any protected side chain infragment PEP.

The diketopiperazine forming dipeptidyl linker and the resultingdiketopiperazine residue comprising C-terminal protecting group comprisethe handle group HG, which makes the cleavage of the peptide PEP fromthe diketopiperazine residue comprising C-terminal protecting grouppossible.

To make this desired function possible, said diketopiperazine formingdipeptidyl linker is constructed in such a way, that the four principalcleavage steps

-   1. the cleavage of each N-terminal protecting group of the amino    acids during the cycles of SPPS,-   2. the cleavage of the fragment C-PEP from the resin by cleaving the    protecting group PG2 from Xaa2, and then cleaving Xaa1 from the    resin by forming the diketopiperazine residue comprising C-terminal    protecting group, and-   3. the cleavage of the diketopiperazine residue comprising    C-terminal protecting group from the peptide PEP, which is the    cleavage of the peptide from HG in the diketopiperazine residue    comprising C-terminal protecting group;-   4. cleavage of any side chain PG;    can be done under different reaction conditions, therefore each    cleavage can be done separately and independently from the other    cleavage at the appropriate point of time in the reaction sequence.

To achieve this function, the chemical nature of the various PGsinvolved in the reaction strategy and the chemical nature of theconnection of the handle group HG of the linker to the peptide is chosenin such a way, that any PG belongs to one of the four types of PGs insuch a way, and that the connection of the handle group HG to thepeptide is cleavable under such reaction conditions, that this groupingof the protecting groups and this selection of the nature of theconnection of the handle group HG to the peptide allow for the desiredand necessary separate and stepwise cleavage.

If there are one or more side chain PGs in the desired peptide C-PEP,one preferred embodiment is, that

-   1. any side chain protecting group is a strong, reductive or mixed    type PG; and-   2. any N-terminal PG of the amino acids used in SPPS in the    synthesis of C-PEP except for the last one, i.e. except for the    N-terminal PG of the N-terminal amino acid of the peptide C-PEP, is    a basic type PG, or-   any N-terminal PG of the amino acids used in SPPS in the synthesis    of C-PEP except for the last one, i.e. except for the N-terminal PG    of the N-terminal amino acid of the peptide C-PEP, is a basic, a    reductive or a mixed type PG, if any side chain protecting group is    not a reductive or mixed type PG; and-   3. PG2 is a weak, a reductive or a mixed type PG, if any side chain    protecting group and any N-terminal PG of the amino acids used in    SPPS in the synthesis of C-PEP except for the last one, i.e. except    for the N-terminal PG of the N-terminal amino acid of the peptide    C-PEP, is not a reductive or mixed type PG, or PG2 is a weak type    PG; and-   4. the N-terminal PG of the last amino acid used in SPPS in the    synthesis of C-PEP, i.e. of the N-terminal amino acid of the peptide    C-PEP, is a basic or a weak type PG, or-   the N-terminal PG of the last amino acid used in SPPS in the    synthesis of C-PEP, i.e. of the N-terminal amino acid of the peptide    C-PEP, is a basic, a weak, a reductive or a mixed type PG, if any    side chain protecting group is not a reductive or mixed type PG; and-   5. the diketopiperazine residue comprising C-terminal protecting    group of PEP or C-PEP is cleavable from the peptide PEP or C-PEP    -   in strong cleaving conditions, or    -   in strong or reductive cleaving conditions, if PG2 and any        N-terminal PG of the amino acids used in SPPS in the synthesis        of C-PEP are not reductive or mixed type PGs, or    -   in weak cleaving conditions, if PG2 and any N-terminal PG of the        amino acids used in SPPS in the synthesis of C-PEP are not weak        type PGs, or    -   in weak 1 cleaving conditions, if PG2 and any N-terminal PG of        the amino acids used    -   in SPPS in the synthesis of C-PEP are not weak 1 type PG.

If there are one or more side chain PGs in the desired peptide C-PEP,one more preferred embodiment is, that

-   1. any side chain protecting group is a strong type PG; and-   2. any N-terminal PG of the amino acids used in SPPS in the    synthesis of C-PEP except for the last one, i.e. except for the    N-terminal PG of the N-terminal amino acid of the peptide C-PEP, is    a basic type PG; and-   3. PG2 is a weak, a reductive or a mixed type PG, if any side chain    protecting group and any N-terminal PG of the amino acids used in    SPPS in the synthesis of C-PEP except for the last one, i.e. except    for the N-terminal PG of the N-terminal amino acid of the peptide    C-PEP, is not a reductive or mixed type PG; and-   4. the N-terminal PG of the last amino acid used in SPPS in the    synthesis of C-PEP, i.e. of the N-terminal amino acid of the peptide    C-PEP, is a basic, a weak, a reductive or a mixed type PG, if any    side chain protecting group is not a reductive or mixed type PG; and-   5. the diketopiperazine residue comprising C-terminal protecting    group of PEP or C-PEP is cleavable from the peptide PEP or C-PEP in    strong cleaving conditions, or    -   in strong or reductive cleaving conditions, if PG2 and any        N-terminal PG of the amino acids used in SPPS in the synthesis        of C-PEP are not reductive or mixed type PGs, or    -   in weak cleaving conditions, if PG2 and any N-terminal PG of the        amino acids used in SPPS in the synthesis of C-PEP are not weak        type PGs, or    -   in weak 1 cleaving conditions, if PG2 and any N-terminal PG of        the amino acids used in SPPS in the synthesis of C-PEP are not        weak 1 type PG.

If there are one or more side chain PGs in the desired peptide C-PEP,another more preferred embodiment is, that

-   1. any side chain protecting group is a strong, reductive or mixed    type PG; and-   2. any N-terminal PG of the amino acids used in SPPS in the    synthesis of C-PEP except for the last one, i.e. except for the    N-terminal PG of the N-terminal amino acid of the peptide C-PEP, is    a basic type PG; and-   3. PG2 is a weak type PG; and-   4. the N-terminal PG of the last amino acid used in SPPS in the    synthesis of C-PEP, i.e. of the N-terminal amino acid of the peptide    C-PEP, is a basic or a weak type PG; and-   5. the diketopiperazine residue comprising C-terminal protecting    group of PEP or C-PEP is cleavable from the peptide PEP or C-PEP    -   in strong or reductive cleaving conditions, if PG2 and any        N-terminal PG of the amino acids used in SPPS in the synthesis        of C-PEP are not reductive or mixed type PGs, or    -   in weak 1 cleaving conditions, if PG2 and any N-terminal PG of        the amino acids used in SPPS in the synthesis of C-PEP are not        weak 1 type PG.

If there are no side chain PGs in the desired peptide C-PEP, onepreferred embodiment is, that

-   1. any N-terminal PG of the amino acids used in SPPS in the    synthesis of C-PEP except for any N-terminal PG of the amino acids    used in SPPS in the synthesis of C-PEP except for the last one, i.e.    except for the N-terminal PG of the N-terminal amino acid of the    peptide C-PEP, is a basic, a reductive or a mixed type PG; and-   2. PG2 is a strong, weak, a reductive or a mixed type PG, if any    N-terminal PG of the amino acids used in SPPS in the synthesis of    C-PEP except for the last one, i.e. except for the N-terminal PG of    the N-terminal amino acid of the peptide C-PEP, is not a reductive    or mixed type PG, or    PG2 is a strong, a weak or a mixed type PG; and-   3. the N-terminal PG of the last amino acid used in SPPS in the    synthesis of C-PEP, i.e. of the N-terminal amino acid of the peptide    C-PEP, is a basic, a strong, a weak, a reductive or a mixed type PG;    and-   4. the diketopiperazine residue comprising C-terminal protecting    group of PEP or C-PEP is cleavable from the peptide PEP or C-PEP    -   in strong cleaving conditions, if PG2 and any N-terminal PG of        the amino acids used in SPPS in the synthesis of C-PEP are not        strong type PGs, or    -   in strong or reductive cleaving conditions, if PG2 and any        N-terminal PG of the amino acids used in SPPS in the synthesis        of C-PEP are not strong, reductive and mixed type PGs, or    -   in weak cleaving conditions, if PG2 and any N-terminal PG of the        amino acids used in SPPS in the synthesis of C-PEP are not        strong or weak type PGs, or    -   in weak 1 cleaving conditions, if PG2 and any N-terminal PG of        the amino acids used in SPPS in the synthesis of C-PEP are not        strong or weak 1 type PGs.

If there are no side chain PG in the desired peptide C-PEP,

one more preferred embodiment is, that

-   1. any N-terminal PG of the amino acids used in SPPS in the    synthesis of C-PEP except for any N-terminal PG of the amino acids    used in SPPS in the synthesis of C-PEP except for the last one, i.e.    except for the N-terminal PG of the N-terminal amino acid of the    peptide C-PEP, is a basic type PG; and-   2. PG2 is a strong, weak, a reductive or a mixed type PG, or-   3. the N-terminal PG of the last amino acid used in SPPS in the    synthesis of C-PEP, i.e. of the N-terminal amino acid of the peptide    C-PEP, is a basic, a strong, a weak, a reductive or a mixed type PG;    and-   4. the diketopiperazine residue comprising C-terminal protecting    group of PEP or C-PEP is cleavable from the peptide PEP or C-PEP    -   in strong cleaving conditions, if PG2 and any N-terminal PG of        the amino acids used in SPPS in the synthesis of C-PEP are not        strong type PGs, or    -   in strong or reductive cleaving conditions, if PG2 and any        N-terminal PG of the amino acids used in SPPS in the synthesis        of C-PEP are not strong, reductive and mixed type PGs, or    -   in weak cleaving conditions, if PG2 and any N-terminal PG of the        amino acids used in SPPS in the synthesis of C-PEP are not        strong or weak type PGs, or    -   in weak 1 cleaving conditions, if PG2 and any N-terminal PG of        the amino acids used in SPPS in the synthesis of C-PEP are not        strong or weak 1 type PGs.

Subject of the invention is a method(C-PEP) for the preparation of apeptide C-PEP,

-   C-PEP comprises a peptidyl radical PEP-C, the C-terminus of PEP-C is    protected by a protecting group DKP-PG, DKP-PG comprises a handle    group HG, optionally a spacer group SG, and a diketopiperazine    residue DKP;-   SG is a spacer group conventionally used in peptide chemistry;-   DKP is a diketopiperazine residue derived from a dipeptide residue    DPR;-   DPR comprises alpha amino acid residues Xaa1 and Xaa2;-   Xaa1 is the C-terminal amino acid residue of DPR;-   Xaa2 is the N-terminal amino acid residue of DPR, and Xaa2 has a    side chain, said side chain is substituted by a functional group FG;-   PEP-C is connected via XaaC⁽¹⁾ to HG;-   XaaC is an amino acid residue of PEP-C;-   index (1) in XaaC⁽¹⁾ denotes the C-terminal position of PEP-C;-   XaaC⁽¹⁾ is the C-terminal amino acid residue of PEP-C;-   HG is a handle group conventionally used in solid phase peptide    synthesis SPPS for connecting the C-terminus of a peptide to the    solid phase, which allows for cleavage of the C-terminus from HG    under conditions, which do not cleave an amide bond connecting two    amino acid residues in a peptide;-   HG is either directly connected to FG, or, if a SG is present, HG is    connected to SG and SG is connected to FG;-   method(C-PEP) comprises a step (iii);-   step (iii) comprises a reaction(INRIFO);-   reaction(INRIFO) is a reaction which comprises an intramolecular    ring formation and a simultaneous cleavage reaction in a peptide    PEP-C-DKP-L-ResinA;-   PEP-C-DKP-L-ResinA is the precursor of C-PEP and comprises PEP-C and    a resin DKP-L-ResinA, with PEP-C being connected to DKP-L-ResinA;-   DKP-L-ResinA comprises a ResinA and a DKP-PG forming linker DKP-L,    with ResinA being connected to DKP-L,-   ResinA is a resin used conventionally as solid phase in SPPS,-   DKP-L comprises HG, optionally SG, and DPR, with the C-terminal    carboxylic acid group of DPR, which is the carboxylic acid group of    Xaa1, being connected to ResinA;-   the intramolecular ring formation in reaction(INRIFO) is a reaction    of the N-terminal amino group of DPR, which is the alpha amino group    of Xaa2, with the C-terminal carboxylic acid group of DPR, thereby    forming DKP, thereby Xaa1 is simultaneously cleaved from ResinA and    DKP-PG is formed;-   HG is chosen in such a way, that the bond between HG and XaaC⁽¹⁾ is    not cleaved during reaction(INRIFO).

By the use of HG, a cleaving site between XaaC⁽¹⁾ and ResinA is providedwhich can be selectively cleaved without cleaving any amide bond betweentwo amino acids in the peptide; the cleaving site being the bond betweenXaaC⁽¹⁾ and HG. By this cleavage, the C-terminus of XaaC⁽¹⁾ is set free,either in form of a unprotected, free carboxylic acid group, or theC-terminal carboxylic acid group is set free in form of an amide group,preferably as C(O)NH₂, depending on the chemical nature of HG.

By the use of HG, this specific DKP comprising C-terminal protectinggroup acts as a conventionally in peptide chemistry used C-terminalprotecting group.

Preferably, PEP-C is prepared prior to the reaction (INRIFO) by a solidphase peptide synthesis SPPS(PEP-C), more preferably the SPPS(PEP-C)uses DKP-L-ResinA as solid phase.

Therefore further subject of the invention is the method(C-PEP), withthe method(C-PEP) as defined above, also with all its preferredembodiments, wherein PEP-C is prepared prior to step (iii) by a solidphase peptides synthesis SPPS(PEP-C), more preferably the SPPS(PEP-C)uses DKP-L-ResinA as solid phase. In SPPS(PEP-C), PEP-C is built bycoupling the XaaC consecutively, first to the solid phase, then to thegrowing peptide chain. The various XaaC can be coupled individually andsequentially, but two or more of them can also be coupled e.g. asdipeptides, tripeptides or oligopeptides to the solid phase or to thegrowing peptide chain.

ResinA is chosen in such a way, that the bond between ResinA and Xaa1 isnot cleaved during SPPS(PEP-C).

Preferably, SPPS(PEP-C) comprises further a step (i) and a step (ii);

-   in step (i) XaaC⁽¹⁾ is attached to DKP-L-ResinA;-   in step (ii) the further amino acids XaaC according to the sequence    of PEP-C are consecutively connected by SPPS(PEP-C) initially to    XaaCW⁽¹⁾ and then to the N-terminus of the growing peptidyl chain,    which is bound via DKP-L to the ResinA;-   HG is chosen in such a way, that the bond between HG and XaaC⁽¹⁾ is    not cleaved during SPPS(PEP-C); and    -   that the bond between HG and XaaC⁽¹⁾ is not cleaved during        reaction(INRIFO);-   with C-PEP, ResinA, DKP-PG, HG, SG, DPR, DKP, Xaa1, XaaC⁽¹⁾, XaaC,    PEP-C, SC-PG, reaction(INRIFO) as defined above, also with all their    preferred embodiments;-   and with the connectivities between PEP-C, HG, SG and DPR and ResinA    as defined above, also with all their preferred embodiments.

Prior to reaction(INRIFO), the N-terminus of DPR, which is alpha aminogroup of Xaa2, is protected by a protecting group PG2.

Therefore further subject of the invention is a method(C-PEP), with themethod(C-PEP) as defined above, also with all its preferred embodiments,wherein step (iii) comprises cleavage of the protecting group PG2;

-   PG2 is an N-terminal protecting group conventionally used in peptide    chemistry and is selected from the group consisting of basic    cleavable type protecting groups, acid cleavable type protecting    groups and reductively cleavable type protecting groups.-   PG2 is cleaved from Xaa2 before the reaction(INRIFO) in step (iii).-   Preferably, PG2 is cleaved from Xaa2 after SPPS(PEP-C).-   Preferably, PG2 is cleaved from Xaa2 after the addition of the    N-terminal amino acid residue of PEP-C in step (ii).-   The cleavage of PG2 from Xaa2 and the reaction(INRIFO) can occur    consecutively or simultaneously.-   PG2 is chosen in such a way, that the bond between PG2 and Xaa2 is    not cleaved during SPPS(PEP-C).-   PG2 and HG are chosen in such a way, that the bond between HG and    XaaC⁾ is not cleaved during the cleavage of PG2 from Xaa2.-   Any side chain of C-PEP or PEP-C can be protected independently from    any other side chain of C-PEP or PEP-C by a protecting group SC-PG,    in case of more than one SC-PG being present in C-PEP or PEP-C,    these SC-PG are independently from each other identical or    different. Any SC-PG is a protecting group which is conventionally    used in peptide chemistry for protecting side chains of amino acid    residues of a peptide or for protecting side chains of amino acids    during SPPS or during HSPPS.-   Preferably, any SC-PG is chosen in such a way, that no SC-PG is    cleaved during SPPS(PEP-C).-   Preferably, any SC-PG is chosen in such a way, that no SC-PG is    cleaved during reaction(INRIFO).-   Preferably, PG2 and any protecting group SC-PG are chosen in such a    way, that no SC-PG is cleaved during the cleavage of PG2 from Xaa2.

In order to avoid complexity of the description, the abbreviation XaaCis used either for the amino acids used to synthesis PEP-C and C-PEP, orit is used for the amino acid residues of PEP-C and C-PEP, or PEP-Nrespectively; and likewise XaaN is used either for the amino acids usedto synthesis PEP-N, or it is used for the amino acid residues of PEP-N.

Therefore these abbreviations do not differentiate between amino acidsand amino acid residues. The skilled person can unambiguouslydistinguish from the context, whether an amino acid or an amino acidresidue is meant.

To summarize the connectivities:

-   PEP-C is connected via XaaC⁽¹⁾ to HG.-   HG is either directly connected via FG to Xaa2, or, if a SG is    present, HG is connected to SG and SG is connected via FG to Xaa2.-   Xaa2 is connected with Xaa1 via a peptide bond, Xaa2 is the    N-terminal and Xaa1 the C-terminal amino acid in DPR.

In PEP-C-DKP-L-ResinA, the carboxylic acid group of Xaa1 is connected toResinA.

-   ResinA is chosen in such a way, that the bond between ResinA and    Xaa1 is not cleaved during SPPS (PEP-C).-   HG is chosen in such a way, that the bond between HG and XaaC⁽¹⁾ is    not cleaved during reaction(INRIFO).-   HG is chosen in such a way, that the bond between HG and XaaC⁽¹⁾ is    not cleaved during SPPS(PEP-C).-   PG2 is chosen in such a way, that the bond between PG2 and Xaa2 is    not cleaved during SPPS(PEP-C).-   PG2 and HG are chosen in such a way, that the bond between HG and    XaaC⁽¹⁾ is not cleaved during the cleavage of PG2 from Xaa2.-   Preferably, any SC-PG is chosen in such a way, that no SC-PG is    cleaved during SPPS(PEP-C).-   Preferably, any SC-PG is chosen in such a way, that no SC-PG is    cleaved during reaction(INRIFO).-   Preferably, PG2 and any protecting group SC-PG are chosen in such a    way, that no SC-PG is cleaved during the cleavage of PG2 from Xaa2.-   Preferably, C-PEP is PEP-C, whose C-terminus is protected by DKP-PG.-   Preferably, DPR consists of the amino acid residues Xaa1 and Xaa2.-   Xaa1 and Xaa2 are chosen in such a way, that they allow the    formation of DKP by reaction(INRIFO).-   Preferably, any side chain of C-PEP is protected by a protecting    group SC-PG-   If any SC-PG is present in C-PEP, then preferably HG is chosen in    such a way, that HG, and thereby DKP-PG, is cleaved from XaaC⁽¹⁾    simultaneously in the reaction which cleaves SC-PG, preferably all    SC-PGs.-   Preferably, SC-PG is selected from the group consisting of basic    cleavable type protecting groups, acid cleavable type protecting    groups and reductively cleavable type protecting groups.-   More preferably, any SC-PG is a strong type PG.-   Preferably, FG, when connected to HG or to SG, is present as a    connecting group CG.-   Preferably, FG is selected from the group consisting of COOH, NH₂,    OH and SH, more preferably consisting of NH₂ and OH; therefore CG is    preferably selected from the group consisting of —C(O)O—, —N(H)—,    —O— and —S—, more preferably consisting of —N(H)—and —O—.-   The bond between HG and FG, or, if a SG is present in DKP-PG, the    bonds between HG and SG and between SG and FG, are chosen to be of    such a chemical nature, that they are not cleaved during SPPS    (PEP-C);    and

that they are not cleaved during reaction(INRIFO), step (i), step (ii)or step (iii); preferably, they are also not cleaved during any cleavageof any protecting group.

Preferably, the bond between HG and FG, or, if a SG is present inDKP-PG, the bonds between HG and SG and between SG and FG, are amide orester bonds, more preferably amide bonds. Especially, these bonds are ofsimilar nature or stability as a conventional amide bond between twoamino acid residues in a peptide.

-   The N-terminus of C-PEP can be protected by a protecting group N-PG,    N-PG is an N-terminal protecting group conventionally used in    peptide chemistry.-   Preferably, N-PG is selected from the group consisting of basic    cleavable type protecting groups, acid cleavable type protecting    groups and reductively cleavable type protecting groups.-   Therefore, C-PEP comprises both the N-terminally unprotected    embodiment and the embodiment, wherein the N-terminus of PEP-C is    protected by N-PG.

Further subject of the invention is a method(C-PEP) for the preparationof C-PEP, characterized by the steps (i), (ii) and (iii), which stepscomprise a solid phase peptides synthesis SPPS(PEP-C) and a subsequentreaction(INRIFO); the SPPS(PEP-C) is done on a resin DKP-L-ResinA assolid support,

-   the DKP-L-ResinA is a ResinA, which carries as a functional group a    DKP-PG forming linker DKP-L,-   DKP-L comprises HG, optionally SG, and DPR, with the Xaa1 of the DPR    being connected via its C-terminal carboxylic acid group to ResinA,-   reaction(INRIFO) is a intramolecular ring formation reaction of the    N-terminal amino group of DPR with the C-terminal carboxylic acid    group of DPR, thereby forming DKP;-   and by reaction(INRIFO) Xaa1 is simultaneously cleaved from ResinA    and DKP-PG is formed;-   in step (i) XaaC⁽¹⁾ is attached to DKP-L-ResinA;-   in step (ii) the further amino acids XaaC according to the sequence    of PEP-C are consecutively connected by SPPS(PEP-C) initially to    XaaC⁽¹⁾ and then to the N-terminus of the growing peptide chain,    which is bound via DKP-L to the ResinA;-   in step (iii), which is done after the addition of the N-terminal    amino acid residue of PEP-C in step (ii), C-PEP is formed by    reaction(INRIFO),-   ResinA is chosen in such a way, that the bond between ResinA and    Xaa1 is not cleaved during SPPS(PEP-C);-   HG is chosen in such a way, that the bond between HG and XaaC⁽¹⁾ is    not cleaved during SPPS(PEP-C); and    -   that the bond between HG and XaaC⁽¹⁾ is not cleaved during        reaction(INRIFO);-   any SC-PG protecting a side chain of C-PEP is chosen in such a way,    that SC-PG is not cleaved during SPPS(PEP-C); and    -   that SC-PG is not cleaved during reaction(INRIFO);-   with C-PEP, ResinA, DKP-PG, HG, SG, DPR, DKP, Xaa1, XaaC⁽¹⁾, XaaC,    PEP-C, SC-PG, reaction(INRIFO) as defined above, also with all their    preferred embodiments;-   and with the connectivities between PEP-C, HG, SG and DPR and ResinA    as defined above, also with all their preferred embodiments.-   Further subject of the invention is a method(DKP-L) for preparation    of a DKP-PG forming linker DKP-L,-   method(DKP-L) comprises a step (DKP-L-i), a step (DKP-L-iii) and    optionally a step (DKP-L-ii);-   in step (DKP-L-i) Xaa2 is coupled to Xaa1;-   in optional step (DKP-L-ii) SG is coupled to Xaa2, if SG is present    in DKP-L;-   in step (DKP-L-iii) HG is coupled either to SG, if SG is present in    DKP-L, or to Xaa2;-   with DKP-PG, DKP-L, DKP, Xaa2, Xaa1, HG and SG as defined above,    also with all their preferred embodiments.-   The steps (DKP-L-i), (DKP-L-iii) and the optional step (DKP-L-ii)    can be done in any order.-   Preferably, at first the step (DKP-L-i) is done, then the optional    step (DKP-L-ii) is done, if SG is present in DKP-L, and the step    (DKP-L-iii) is done as the last step.

Further subject of the invention is a method(DKP-L-ResinA) forpreparation of DKP-L-ResinA,

-   method(DKP-L-ResinA) is a method(X1) or a method(X2);-   method(X1) comprises a step (X1-i), a step (X1-ii), a step (X1-iv)    and optionally a step (X1-iii);    -   in step (X1-i) the amino acid Xaa1 is coupled to ResinA;    -   in step (X1-ii) the amino acid Xaa2 is coupled to Xaa1;    -   in the optional step (X1-iii) SG is coupled to the side chain of        Xaa2, if SG is present in DKP-L-ResinA;    -   in step (X1-iv) HG is coupled either to SG, if SG is present in        DKP-L-ResinA, or to Xaa2;-   method(X2) comprises a step (X2-i);    -   in step (X2-i) DPK-L is coupled to ResinA;-   with DKP-L-ResinA, ResinA, DKP-PG, DKP-L, DKP, Xaa2, Xaa1, HG and SG    as defined above, also with all their preferred embodiments.-   In method (X1), the steps (X1-i), (X1-ii), (X1-iv) and the optional    step (X1-iii) can be done in any order.-   Preferably, at first the step (X1-i), then the step (X1-ii) is done,    then the optional step (X1-iiii) is done, if SG is present in    DKP-L-ResinA, and the step (X1-iv) is done as the last step.

HG, any SG, Xaa2 and Xaa1, when used as building blocks inmethod(DKP-L-ResinA) or in method(DKP-L), can carry a protecting group:

-   Xaa1, used as building block in method(XI) or method(DKP-L), is used    as a conventionally C-terminally protected amino acid, the    protecting group being a protecting group C-PG. The alpha amino    group of Xaa1 is unprotected and is the coupling site in the    respective coupling reaction.-   Xaa2, used as building block in method(X1) or method(DKP-L), is used    as a conventionally N-terminally protected amino acid, the    protecting group being a protecting group N-PG. The 1-carboxylic    acid group of Xaa2 is unprotected and is the coupling sites in the    respective coupling reaction.

Any side chain of Xaa1 or Xaa2 is preferably also protected by a SC-PG.

-   C-PG is a protecting group conventionally used in peptide chemistry    for protecting the carboxylic acid group of an amino acid or for    protecting the C-terminus of a peptide.-   Preferably, C-PG is selected from the group consisting of basic    cleavable type protecting groups, acid cleavable type protecting    groups and reductively cleavable type protecting groups.-   Each HG and SG, in form of individual building blocks used in the    respective coupling reactions, has at least two reactive functional    groups. The first reactive functional group is used as a    functionality resembling the alpha amino group of an amino acid    building block in peptide synthesis and can be protected by a    suitable protecting group, preferably by a protecting group N-PG;    preferably, this functional group is OH or NH₂ and is present in the    protected state as —O— or —N(H)—.    -   The other reactive functional group of HG and SG is used as a        functionality resembling the carboxylic acid group of an amino        acid building block in peptide synthesis and is usually        unprotected and is the coupling site in the respective coupling        reaction. Preferably, this unprotected site is a carboxylic acid        group. After this coupling reaction, any protecting group of the        first reactive functional group, preferably said N-PG, can be        cleaved in order to make this first functional group available        for the next coupling reaction.

The DKP-PG forming linker DKP-L, obtainable by method(DKP-L), usuallystill carries any protecting group of HG in order to be usable in thecoupling to ResinA in method(X2). Prior to the coupling in method(X2), aC-PG of Xaa1 must be cleaved off. Preferably, method(DKP-L) comprisesthis cleaving of C-PG from Xaa1. Therefore, DKP-L comprises bothembodiments, one embodiment with a protecting group C-PG on Xaa1, theother embodiment without a protecting group C-PG on Xaa1.

In DKP-L-ResinA, HG can still carry a protecting group which was presentin the building block HG used for preparing DKP-L-ResinA. Any protectinggroup on HG must be cleaved prior to step (i) in method(C-PEP).Preferably, method(DKP-L-ResinA) comprises this cleaving of anyprotecting group from HG. Therefore, DKP-L-ResinA comprises bothembodiments, one with any protecting group on HG, the other without anyprotecting group on HG.

Further subject of the invention is a method(PEP-HSPPS) for thepreparation of a peptide PEP,

-   method(PEP-HSPPS) comprises a step (i-pep) and a step (ii-pep),-   in step (i-pep) a peptide C-PEP is prepared according to    method(C-PEP); then-   in step (ii-pep)C-PEP obtained in step (i-PEP) is coupled with an    N-terminally protected amino acid or with an N-terminally protected    peptide PEP-N by homogeneous solution phase peptide synthesis HSPPS;-   with method(C-PEP), C-PEP and PEP-N being as defined above, also    with all its preferred embodiments.-   Method(PEP-HSPPS) is a method(C-PEP) comprising the further step    (ii-pep).-   Any side chain of PEP-N can be protected independently from any    other side chain of PEP-N by a protecting group SC-PG, in case of    more than one SC-PG being present in PEP-N, these SC-PG are    independently from each other identical or different; with SC-PG    being as defined above, also with all its preferred embodiment.

C-PEP in method(PEP-HSPPS) is used N-terminally unprotected. Therefore,any protecting group N-PG, which protects the N-terminus of C-PEP, iscleaved prior to the coupling reaction of method(PEP-HSPPS). Thiscleaving reaction is preferably comprised in method(C-PEP). Since PEP-Cis made by SPPS(PEP-C), the N-terminal amino acid of PEP-C used inSPPS(C-PEP) is usually used with a protected amino group N-PG on itsalpha amino group. Depending on the nature of this protecting group N-PGof the N-terminus of PEP-C this N-PG can be cleaved from the N-terminussimultaneously under the condition of the ring formation inreaction(INRIFO), or it can be cleaved from the N-terminussimultaneously with the cleaving of PG2 from Xaa2 prior to the reaction(INRIFO).

Further subject of the invention are following methods:

-   1. a method(PEP-HSPPS), wherein    -   the DKP-L-ResinA of method(C-PEP) has been prepared by the        method(DKP-L-ResinA);-   2. a method(PEP-HSPPS), wherein    -   the DKP-L-ResinA of method(C-PEP) has been prepared by        method(X1) of the method(DKP-L-ResinA);-   3. a method(PEP-HSPPS), wherein    -   the DKP-L-ResinA of method(C-PEP) has been prepared by        method(X2) of the method(DKP-L-ResinA); and wherein    -   the DKP-L of method(DKP-L-ResinA) has been prepared by the        method(DKP-L);-   4. a method(C-PEP), wherein    -   the DKP-L-ResinA has been prepared by the method(DKP-L-ResinA);-   5. a method(C-PEP), wherein    -   the DKP-L-ResinA has been prepared by method(X1) of the        method(DKP-L-ResinA);-   6. a method(C-PEP), wherein    -   the DKP-L-ResinA has been prepared by method(X2) of the        method(DKP-L-ResinA); and wherein    -   the DKP-L of method(DKP-L-ResinA) has been prepared by the        method(DKP-L).

Further subject of the invention is a compound selected from the groupconsisting of C-PEP, PEP-C-DKP-L-ResinA, DKP-L-ResinA and DKP-L, withC-PEP, PEP-C-DKP-L-ResinA, DKP-L-ResinA and DKP-L as defined above, alsowith all their preferred embodiments.

Further subject of the invention is the use of C-PEP, with C-PEP beingas defined above, also with all its preferred embodiments, inhomogeneous solution phase peptide synthesis HSPPS for the preparationof a peptide PEP by a coupling reaction of C-PEP with an N-terminallyprotected amino acid or with an N-terminally protected peptide PEP-N.

-   Further subject of the invention is the use of a compound selected    from the group consisting of C-PEP, PEP-C-DKP-L-ResinA, DKP-L-ResinA    and DKP-L; or the use of DKP-L as a DKP-PG forming linker,-   in peptide chemistry; or-   for the preparation of a peptide; or-   in a method for the preparation of a peptide; or-   in a step of a method for the preparation of a peptide; or-   in a peptide coupling reaction; or-   in SPPS for the preparation of a peptide; or-   in HSPPS for the preparation of a peptide;-   with C-PEP, PEP-C-DKP-L-ResinA, DKP-L-ResinA, DKP-L and DKP-PG as    defined above, also with all their preferred embodiments.

Any of the following embodiments of the invention are comprised in thehitherto described embodiments of the invention.

Further subject of the invention is a method(A) for the preparation of acompound of formula (III-PEP-PG)

-   by homogenous solution phase coupling of-   an amino acid, which is N-terminally protected by a protecting group    PGIII, or of-   an N-terminally protected peptide fragment PEP-N, the N-terminally    protected peptide fragment PEP-N being a compound of formula    (III-PEP—N-PG),

PGIII-(XaaN^((ipn)))_(pn)  (III-PEP—N-PG)

with a compound of formula (III-H);

-   HG is a handle group conventionally used in solid phase peptide    synthesis SPPS for connecting the C-terminus of a peptide to the    solid phase, which allows for cleavage of the C-terminus from HG    under conditions, which do not cleave an amide bond connecting two    amino acid residues in a peptide;-   n is 0 or 1;-   SG is a spacer group conventionally used in peptide chemistry;-   Xaa1 is an alpha amino acid residue;-   Xaa2 is a 2-(C₁₋₅ alkyl)-alpha amino acid residue, wherein the C₁₋₅    alkyl group is substituted by a functional group FG, FG is selected    from the group consisting NH₂, OH, SH and COOH; FG is bonded with SG    when n is 1; FG is bonded to HG when n is 0, and therefore FG is    present in the compound of formula (III-PEP-PG) as a connecting    group CG selected from the group consisting —N(H)—, —O—, —S— and    —C(O)O—;-   PEP-C is a peptidyl radical of formula (XaaC^((ipc)))_(pc);-   the hydrogen H denoted with (1) in formula (III-H) is a hydrogen of    the unprotected N-terminus of PEP-C;-   XaaC is an amino acid residue of the peptidyl radical PEP-C;-   in XaaC^((ipc)), (ipc) signifies the index of XaaC of PEP-C at the    position ipc, with the position count starting from the C-terminus    of PEP-C,-   pmax is 502;-   pc is an integer of from 2 to (pmax-2) and represents the total    number of amino acid residues in PEP-C;-   ipc is an integer of from 1 to pc;-   PGIII in formulae (III-PEP-PG) and (III-PEP-N-PG) are identical and    is an N-terminal protecting group commonly used in peptide    chemistry;-   PEP is a peptidyl radical of formula (XaaP^((ip)))_(p);-   pn is an integer of from 2 to (pmax-pc) and represents the total    number of amino acid residues in PEP-N;-   p is pc+pn;-   XaaN is an amino acid residues of the peptide fragment PEP-N;-   in XaaN^((ipn)), (ipn) signifies the index of XaaN of PEP-N at the    position ipn, with the position count starting from the C-terminus    of PEP-N;-   XaaP is an amino acid residue;-   in XaaP^((ip)), (ip) signifies the index of XaaP of PEP at the    position ip, with the position count starting from the C-terminus of    PEP;-   ipn is an integer of from 1 to pn;-   ip is an integer of from 1 to p;-   with the proviso, that XaaP^((ip)) is identical with XaaC^((ipc))    for ip having a value from 1 to ipc; and XaaP^((ip)) is identical    with XaaN^((ipn)) for ip having the value (pc+ipn);-   with pmax, pc, XaaC, XaaC^((ipc)), ipc and compound of formula    (III-H) being as defined above, also with all their preferred    embodiments;-   XaaC in formula (III-H) and XaaN are independently from each other    identical or different.

Therefore, PEP-C is a peptidyl radical having pc amino acid residuesXaaC.

Preferably, the alpha amino group of Xaa1 is coupled to the 1-carboxygroup of Xaa2 by a peptide bond.

Compound of formula (III-H) is an embodiment of the above defined C-PEP.HG, SG, Xaa2 and Xaa1 are embodiments of the respective HG, SG, Xaa2 andXaa1 of the above defined peptide C-PEP.

-   The cyclic dipeptide in e.g. formula (III-H) is one embodiment of    the above mentioned DKP, i.e. the diketopiperazine residue derived    from DPR.

Preferably, PEP-C is prepared by SPPS.

The SPPS, by which PEP-C is prepared, is called above SPPS(PEP-C).

Preferably, PEP-C is a peptidyl radical of formula (XaaC^((ipc)) _(pc),which has been synthesized by SPPS using amino acids of formulaPGXaaC^((ipc))-XaaC^((ipc))-OH.

-   PGXaaC is an N-terminal protecting group conventionally used in SPPS    and is selected from the group consisting of basic cleavable type    protecting groups, acid cleavable type protecting groups and    reductively cleavable type protecting groups.-   In PGXaaC^((ipc)), the index (ipc) defines PGXaaC^((ipc)) as the    protecting group of the amino acid PGXaaC^((ipc))-XaaC^((ipc))—OH,    with each PGXaaC^((ipc)) being independently from each other    identical or different from another PGXaaC^((ipc)).

Preferably, PGXaaC and PGXaaC(^(ipc)) respectively is an N-terminalprotecting group conventionally used in SPPS to protect the alpha aminogroup of any amino acid PGXaaC-XaaC-OH andPGXaaC^((ipc))-XaaC^((ipc))-OH respectively used in the SPPS tosynthesize PEP-C.

In order to avoid complexity of the description, the abbreviationsPGXaaC in the text is used either for the protecting group of the aminogroup of the amino acids used to synthesis PEP-C and C-PEP, or it isused for the N-terminal protecting group of the N-terminal amino acidresidues of PEP-C and C-PEP at the various stages during SPPS. Theskilled person can unambiguously distinguish from the context, which ofthe two meanings is meant.

Therefore, PEP-N is a peptidyl radical having pn amino acid residuesXaaN.

Therefore, PEP is a peptidyl radical having p amino acid residues XaaP.

The residue of formula (III-res)

-   which appears e.g. in the formulae (III-PEP-PG) and (III-H), is an    embodiment of the DKP-PG mentioned above;-   with HG, SG, n, Xaa2 and Xaa1 being as defined above;-   with Xaa2 and Xaa1 forming the DKP mentioned above;-   and with (8) denoting the bond between the peptidyl radical PEP-C    and HG.

Compound of formula (III-PEP-PG) is an embodiment of above definedpeptide PEP and can also be an embodiment of above defined C-PEP.

PEP-N can be prepared by conventional peptide synthesis, either by SPPS,by HSPPS or by a combination of SPPS and HSPPS, preferably by SPPS.

In case that the compound of formula (III-PEP-PG) shall be used in anext HSPPS coupling according to method(A) as a next fragment C-PEP witha next fragment PEP-N, only the N-terminal protecting group PGIII ofsaid compound of formula (III-PEP-PG) needs to be removed, to providefor said next fragment C-PEP, i.e. for the next compound of formula(III-H), for said next HSPPS coupling according to method(A).

Since both the compound of formula (III-H) and PEP-N may themselves havebeen prepared by method(A) in one of their preparation steps, they canhave practically any number of amino acids as long as the solution phasecoupling still works in a reasonable time.

Preferably, pmax is 500, more preferably pmax is 400 or 402, even morepreferably 300 or 302, especially 200 or 202, more especially 150 or152, even more especially 100 or 102, particularly 80 or 82, moreparticularly 50 or 52, even more particularly 25 or 27.

Preferably, the peptidyl radical PEP-C is a linear peptidyl radical, andpreferably PEP-N is a linear peptide, resulting in a linear peptidylradical PEP.

Preferably, if PEP-C in compound of formula (III-H) and/or PEP-N havebeen prepared using SPPS, they have independently from each other offrom 2 to 100, more preferably of from 2 to 50, even more preferably offrom 2 to 40, especially preferably of from 2 to 25 amino acid residues.

Preferably, if PEP-C in compound of formula (III-H) and/or PEP-N havebeen prepared using HSPPS, they have independently from each other fromof 2 to 250, more preferably of from 2 to 200, even more preferably offrom 2 to 100, especially preferably of from 2 to 50, in particular offrom 2 to 25 amino acid residues.

-   Any functional groups on the side chains of the individual amino    acid residues of peptidyl radical PEP-C and of PEP-N are    independently from each protected or unprotected by protecting    groups SC-PG;-   preferably, all functional groups on the side chains of the    individual amino acid residues of peptidyl radical PEP-C and of    PEP-N are protected by protecting groups SC-PG or unprotected;-   more preferably, all functional groups on the side chains of the    individual amino acid residues of peptidyl radical PEP-C and of    PEP-N are protected during the solution phase coupling according to    method(A) of fragment PEP-N with compound of formula (III-H),-   even more preferably, all functional groups on the side chains of    the individual amino acid residues of peptidyl radical PEP-C and of    PEP-N are protected by strong type PG.-   Preferably, the C terminus or, in case that the C-terminal amino    acid residue has a side chain, the side chain of the C-terminal    amino acid residue of the peptidyl radical PEP or PEP-C    respectively, is bonded to HG;-   more preferably, the C terminus of the peptidyl radical PEP or PEP-C    respectively, is bonded to HG.

Preferably,

-   HG is a handle group conventionally used in solid phase peptide    synthesis SPPS to connect an amino acid, which will become the    C-terminal amino acid residue of a peptide, which is to be    synthesised by SPPS, via said HG to a solid phase, preferably to a    ResinA.-   HG allows for cleavage of the C-terminal amino acid residue from HG    under conditions, which do not cleave an amide bond connecting two    amino acid residues in a peptide.

More preferably,

-   HG is a handle group selected from the group consisting of handle    group of formula (HGF-I), handle group of formula (HGF-II), handle    group of formula (HGF-III), handle group of formula (HGF-IV), handle    group of formula (HGF-V) and handle group of formula (HGF-VI),

wherein

-   (*) denotes the bond between the C atom of the C terminus of the    respective peptidyl radical, e.g. of PEP for formulae (III-PEP-PG),    of PEP-C for formula (III-H) or of PEP-C in method (C-PEP), and HG,-   or denotes, in case that the C-terminal amino acid residue of the    respective peptidyl radical, e.g. of PEP for formulae (III-PEP-PG),    of PEP-C for formula (III-H) or of PEP-C in method(C-PEP), has a    side chain and is connected via this side chain to HG, the bond    between the side chain of the C terminal amino acid residue of the    respective peptidyl radical, e.g. of PEP for formulae (III-PEP-PG),    of PEP-C for formula (III-H) or of PEP-C in method(C-PEP), and HG,-   (**) denotes the bond between HG and SG when n is 1, or denotes the    bond between HG and FG when n is 0, with SG and FG as defined above,    also with all their preferred embodiments;-   R1, R2, R3, R4, R10 and R11 are identical or different and    independently from each other selected from the group consisting of    hydrogen and O—C₁₋₄ alkyl,-   s1-1, s2, s3, s4 and s6 are identical or different and independently    from each other selected from the group consisting of 1, 2, 3 and 4,-   s5-1 is 0, 1, 2, 3 or 4,-   s1-2, s5-2 and s5-3 are identical or different and independently    from each other 0 or 1,-   T1-1 is O or NH,-   T1-2 and T5-1 are O,-   with n, SG, FG, PEP-C and method(C-PEP) as defined above, also with    all their preferred embodiments.

Preferably,

-   (*) denotes the bond between the C atom of the C terminus of the    respective peptidyl radicals, e.g. of PEP for formulae (III-PEP-PG),    of PEP-C for formula (III-H) or of PEP-C in method(C-PEP), and HG.

Preferably,

-   SG is a spacer group conventionally used in SPPS, preferably    comprising one or more, more preferably 1 to 500, ethylenoxide    units.

More preferably,

-   SG is a spacer group selected from the group consisting of spacer    group of formula (SG-I), spacer group of formula (SG-II), spacer    group of formula (SG-III), spacer group of formula (SG-IV) and    spacer group of formula (SG-V);

m1, m5, m6, m7, m9, m10, m11 and m12 are identical or different andindependently from each other an integer of 1 to 500;

-   m2, m3 and m4 are identical or different and independently from each    other 1, 2, 3 or 4,-   (***) is the bond from SG to HG when n is 1,-   (****) is the bond between SG and Xaa2 when n is 1.-   (***) is the bond denoted by (**) in the respective embodiments of    HG, when n is 1.-   (****) is the bond between SG and FG, when n is 1;-   with HG, Xaa2 and n as defined above, also with all their preferred    embodiments.-   Preferably, XaaC and XaaN are alpha amino acid residues.-   More preferably, XaaC and XaaN are naturally occurring alpha amino    acid residues.-   If XaaC or XaaN carries a side chain with a functional group, this    functional group of the side chain of XaaC or XaaN is either    protected or unprotected, preferably it is protected.-   More preferably, XaaC and XaaN are identical or different and are    independently from each other selected from the group consisting of    Ala, Aib, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,    Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr, Asp, Asn, Glu and Gln; where    any functional group in the side chain is either protected or    unprotected, preferably protected.-   Preferably, PGIII is selected from the group consisting of basic    type PGs, strong type PGs, weak type PGs and reductive type PGs.-   If all functional groups on the side chains of the individual amino    acid residues of peptidyl radical PEP-C and of PEP-N are protected    by strong acid cleavable type protecting groups, and in case that    the compound of formula (III-PEP-PG) shall be used in a next HSPPS    coupling according to method(A) as a next fragment C-PEP with a next    fragment PEP-N, then PGIII is preferably selected from the group    consisting of basic type PGs, weak type PGs and reductive type PGs.-   Handle groups of formula (HGF-I) and handle groups of formula    (HGF-IV) are cleavable from the PEP-C by strong cleaving conditions,    handle groups of formula (HGF-II) are cleavable by strong cleaving    conditions,    handle groups of formula (HGF-III) are cleavable by weak or by    strong cleaving conditions,    handle groups of formula (HGF-V) are cleavable by reductive cleaving    conditions, and    handle groups of formula (HGF-VI) are cleavable by weak or by strong    cleaving conditions-   Preferably, HG is a handle group selected from the group consisting    of handle group of formula (HGF-I), handle group of formula (HGF-IV)    and handle group of formula (HGF-VI).-   Preferably, R1, R2, R3, R4, R10 and RI 1 are identical or different    and independently from each other selected from the group consisting    of hydrogen and O—CH₃.-   More preferably, R1 and R2 are identical and selected from the group    consisting of hydrogen and O—CH₃.-   More preferably, R3, R4, R10 and R11 are O—CH₃.    Preferably, s1-1 and s6 are 1.    Preferably, s1-2, s5-1, s5-2 and s5-3 are independently from each    other 0 or 1.

Preferably, s2 and s3 are 4. Preferably, s4 is 1 or 2.

Preferably, T1-1 is NH, s1-1 is 1 and s1-2 is 1.Preferably, T1-1 is O, s1-1 is 1 and s1-2 is 0.Preferably, T1-1 is O, s1-1 is 1 and s1-2 is 1.

Especially, HG is a handle group selected from the group consisting ofhandle group of formula (HG-Ia), handle group of formula (HG-Ib), handlegroup of formula (HG-Ic), handle group of formula (HG-Id), handle groupof formula (HG-II), handle group of formula (HG-III), handle group offormula (HG-IVa), handle group of formula (HG-IVb), handle group offormula (HG-Va), handle group of formula (HG-Vb) and handle group offormula (HG-VI),

wherein(*) is as defined above, also with all its preferred embodiments,(**) is as defined above, also with all its preferred embodiments.

More especially, HG is a handle group selected from the group consistingof handle group of formula (HG-Ia), handle group of formula (HG-Ib),handle group of formula (HG-Ic), handle group of formula (HG-Id), handlegroup of formula (HG-IVa), handle group of formula (HG-IVb), and handlegroup of formula (HG-VI).

-   Even more especially, HG is a handle group of formula (HG-Ia), a    handle group of formula (HG-IVa) or a handle group of formula    (HG-VI).

The various handle groups HG are known handle groups or are structurallyclosely related derivatives of known handle groups. The reactionconditions necessary for cleaving any of these handle groups HG from apeptidyl radical connected to the respective handle group HG, are alsoknown in peptide chemistry.

The handle group of formula (HG-Ia) is derived from the Rink amidehandle, (HG-Ib), (HG-Ic) and (HG-Id) from benzhydryl handles, (HG-II)from the PAL handle, (HG-III) from the Sieber handle, (HG-IVa) from theHMPA(-Wang) handle, (HG-IVb) from the HMPP(-Wang) handle, and (HG-Va)and (HG-Vb) from allyl handles, (HG-VI) from Ramage handle.

-   Preferably, m1, m5, m6, m7, m9, m10, m11 and m12 are identical or    different and independently from each other 1, 2, 3, 4, 5, 6, 7, 8,    9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,    26, 27, 28, 29, 30;-   more preferably, m1, m5, m6, m7, m9, m10, m11 and m12 are identical    or different and independently from each other 1, 2, 3, 4, 5, 6, 7,    8, 9, 10, 11, 12, 23 or 27;-   even more preferably m1, m5, m6, m7, m10, m11 and m12 are identical    or different and independently from each other 1, 2, 3, 4, 5, 6, 7,    8, 9 or 10;    -   m9 is 4, 8, 12 or 27;-   especially m1 is 3; m5 is 1 or 2; m6 and m7 are 2; m9 is 4, 8, 12 or    27; m10 is 1; m11 is 3.-   Xaa1 is preferably selected from the group consisting of    non-naturally occurring alpha amino acids, naturally occurring alpha    amino acid residues;-   more preferably selected from the group consisting of naturally    occurring alpha amino acid residues, alpha-N-methylamino acid    residues, L-Hpr residue, D-Hpr residue, DL-Hpr residue,    2-(C₁₋₅-alkyl)-D-amino acid residues, 2-(C₁₋₅-alkyl)-L-amino acid    residues, 2-(C₁₋₅-alkyl)-DL-amino acid residue and a residue derived    from compound of formula (HypX);

wherein

X is O, S or C(R13)R14;

R5, R7, R12, R13 and R14 are identical or different and independentlyfrom each other selected from the group consisting of hydrogen, C₁₋₄alkyl and O-R8;

-   R8 is a protecting group conventionally used for side chain    protection in peptide chemistry, or a substituent of formula    (Sub-R8);

whereinm8 is 1, 2,3,4,5,6,7,8,9 or 10;R9 is C₁₋₄ alkyl.

Preferably,

-   X is C(R13)R14;-   R5, R7, R12 and R14 are hydrogen;-   R13 is O—R8;-   R8 is a protecting group conventionally used for side chain    protection in peptide chemistry.

The alpha-N-methylamino acid residues is preferably selected from thegroup consisting of L-alpha-N-methylamino acid residues,D-alpha-N-methylamino acid residues and DL-alpha-N-methylamino acidresidues;

-   more preferably selected from the group consisting of    N-methylglycine residue (sarcosine), L-N-methylphenylalanine    residue, D-N-methylphenylalanine residue, DL-N-methylphenylalanine    residue, L-N-methylalanine residue, D-N-methylalanine residue,    DL-N-methylalanine residue, L-N-methylvaline residue,    D-N-methylvaline residue, DL-N-methylvaline residue,    L-N-methyltryptophane residue, D-N-methyltryptophane residue,    DL-N-methyltryptophane residue.

The naturally occurring alpha amino acid residue is preferably selectedfrom the group consisting of Pro residue and Gly residue; morepreferably selected from the group consisting of L-Pro residue, D-Proresidue, DL-Pro residue and Gly residue.

Preferably, compound of formula (HypX) is derived from L-Hyp, D-Hyp orDL-Hyp, more preferably from L-4Hyp, D-4Hyp or DL-4Hyp.

Especially, Xaa1 is selected from the group consisting ofL-N-methylglycine residue, D-N-methylglycine residue, DL-N-methylglycineresidue, L-N-methylphenylalanine residue, D-N-methylphenylalanineresidue, DL-N-methylphenylalanine residue, L-Pro residue, D-Pro residue,DL-Pro residue, side chain protected L-Hyp residue, side chain protectedD-Hyp residue, side chain protected DL-Hyp residue, L-Hpr residue, D-Hprresidue and DL-Hpr residue; with Hyp being preferably 4Hyp.

More especially, Xaa1 is L-N-methylphenylalanine residue,D-N-methylphenylalanine residue, DL-N-methylphenylalanine residue, L-Proresidue, D-Pro residue, DL-Pro residue, side chain protected L-Hypresidue, side chain protected D-Hyp residue, side chain protected DL-Hypresidue; with Hyp being preferably 4Hyp.

Even more especially, Xaa1 is D-Pro residue, D-N-methylphenylalanineresidue or side chain protected D-Hyp residue; with Hyp being preferably4Hyp.

FG is bonded with SG via the bond (***) in the respective embodiments ofSG, when n is 1, or FG is bonded to HG via the bond (**) in therespective embodiments of HG.

Preferably, Xaa2 is selected from the group consisting of L-Lys residue,D-Lys residue, DL-Lys residue, L-Orn residue, D-Orn residue, DL-Ornresidue, L-4-aminoproline residue, D-4-aminoproline residue,DL-4-aminoproline residue, L-alpha,gamma-diamino

butanoic acid residue, D-alpha,gamma-diaminobutanoic acid residue,DL-alpha,gamma-diamino

butanoic acid residue, L-alpha,beta-diaminopropanoic acid residue,D-alpha,beta-diamino

propanoic acid residue, DL-alpha,beta-diaminopropanoic acid residue,L-Ser residue, D-Ser residue, DL-Ser residue, L-Thr residue, D-Thrresidue, DL-Thr residue, L-Cys residue, D-Cys residue, DL-Cys residue,L-homocysteine residue, D-homocysteine residue, DL-homocysteine residue,L-Asp residue, D-Asp residue, DL-Asp residue, L-Glu residue, D-Gluresidue and DL-Glu residue.

Preferably, FG is NH₂ or OH, more preferably NH₂; therefore CG ispreferably —N(H)— or —O—, more preferably —N(H)—; therefore Xaa2 ispreferably selected accordingly.

-   More preferably, Xaa2 is selected from the group consisting of L-Lys    residue, D-Lys residue, DL-Lys residue, L-alpha,beta-diamino    propanoic acid residue, D-alpha,beta-diamino    propanoic acid residue and DL-alpha,beta-diamino    propanoic acid residue.-   Even more preferably, Xaa₂ is L-Lys residue or L-alpha,beta-diamino    propanoic acid residue.-   A preferred embodiment is the combination, wherein the Xaa2 is an    L-alpha amino acid residue and Xaa1 is a D-alpha amino acid residue,    or alternatively Xaa2 is a D- and Xaa1 is a L-alpha-amino acid    residue, with Xaa1 and Xaa2 as defined above, also with all their    preferred embodiments.-   More preferably, Xaa1 is selected from the group consisting of L-Pro    residue, D-Pro residue, DL-Pro residue, L-N-methylphenylalanine    residue, D-N-methylphenylalanine residue and    DL-N-methylphenylalanine residue; and Xaa2 is selected from the    group consisting of L-Lys residue, D-Lys residue, DL-Lys,    L-alpha,beta-diamino    propanoic acid residue, D-alpha,bcta-diaminopropanoic acid residue    and DL-alpha,beta-diamino    propanoic acid residue.-   Even more preferably, Xaa1 is D-Pro or D-N-methylphenylalanine    residue, and Xaa2 is L-Lys or L-alpha,beta-diamino    propanoic acid residue; or Xaa1 is L-Pro or L-N-methylphenylalanine    residue, and Xaa2 is D-Lys or D-alpha,beta-diamino    propanoic acid residue.

Especially, Xaa2 is of L- and Xaa1 is of D-configuration, with Xaa1 andXaa2 as defined above, also with all their preferred embodiments.

More especially, Xaa1 is D-Pro or D-N-methylphenylalanine residue, andXaa2 is L-Lys or L-alpha,beta-diamino

propanoic acid residue.

The homogenous solution phase coupling in method(A), i.e. HSPPS, iscarried out using conventional process parameters and reagents typicalfor HSPPS.

HSPPS is conventionally done in a solvent and using one or more couplingreagents, and is done preferably in the presence of one or more couplingadditives, and preferably in the presence of one or more tertiary bases.

Preferable coupling reagents used in HSPPS are phoshonium or uroniumsalts and carbodiimide coupling reagents.

Phosphonium and uronium salts are preferably derivatives ofbenzotriazol; more preferably Phosphonium and uronium salts are selectedfrom the group consisting of

-   BOP (Benzotriazole-l-yl-oxy-tris-(dimethyl amino)-phosphonium    hexafluorophosphate),-   PyBOP (Benzotriazol-1-yl-oxy-trispyrrolidinophosphonium    hexafluorophosphate),-   HBTU (O-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate),-   HCTU (O-(1H-6-chloro-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate),-   TCTU (O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium    tetrafluoroborate),-   HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate),-   TATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    tetrafluoroborate),-   TBTU (O-(benzotriazol-1-yl)-1,1,3,3-tetra    methyluronium tetrafluoroborate),-   TOTU    (O-[cyano(ethoxycarbonyl)methyleneamino]-1,1,3,3-tetramethyluronium    tetrafluoroborate),-   HAPyU (O-(benzotriazol-1-yl)oxybis-(pyrrolidino)-uronium    hexafluorophosphate,-   PyAOP (Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium    hexafluorophosphate),-   COMU    (1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]methanaminiumhexafluorophosphate),-   PyClock    (6-chloro-benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium    hexafluorophosphate),-   PyOxP    (O-[(1-cyano-2-ethoxy-2-oxoethylidene)amino]-oxytri(pyrrolidin-l-yl)    phosphonium hexafluorophosphate) and-   PyOxB    (O-[(1-cyano-2-ethoxy-2-oxoethylidene)amino]-oxytri(pyrrolidin-1-yl)    phosphonium tetrafluoroborate).

Carbodiimide coupling reagents are preferably selected from the groupconsisting of diisopropyl-carbodiimide (DIC), dicyclohexyl-carbodiimide(DCC) and water-soluble carbodiimides (WSCDI) such as1-ethyl-3-(3-dimethylaminopropyl)

carbo

diimide (EDC)

Other coupling techniques use pre-formed active esters, such ashydroxysuccinimide (HOSu) and p-nitrophenol (HONp) esters, pre-formedsymmetrical anhydrides, non-symmetrical anhydrides such asN-carboxyanhydrides (NCAs) and acid halides, such as acyl fluoride oracyl chloride.

Preferred coupling reagents are phoshonium or uronium coupling reagents,especially TCTU, TOTU or PyBop.

Preferably, the conjugated acid of said tertiary base used in HSPPS hasa pKa value of from 7.5 to 15, more preferably of from 7.5 to 10. Saidtertiary base is preferably trialkylamines, such asdiisopropylethylamine (DIEA) or triethylamine (TEA), furtherN,N′-di-C₁₋₄alkylanilines, such as N,N-diethylaniline, 2,4,6-tri-C₁₋₄alkylpyridines, such as collidine (2,4,6-trimethylpyridine), or N—C₁₋₄alkyl-morpholines, such as N-methylmorpholine, with any C₁₋₄ alkyl beingidentical or different and independently from each other straight orbranched C₁₋₄ alkyl.

A coupling additive is preferably a nucleophilic hydroxy compoundcapable of forming activated esters, more preferably having an acidic,nucleophilic N-hydroxy function wherein N is imide or is N-acyl orN-aryl substituted triazeno, the triazeno type coupling additive beingpreferably a N-hydroxy-benzotriazol derivative (or1-hydroxy-benzotriazol derivative) or a N-hydroxybenzotriazinederivative. Such coupling additives have been described in WO 94/07910and EP 410 182. Since they also act as scavengers, they are also calledscavengers.

Preferred coupling additives are selected from the group consisting ofN-hydroxy-succinimide (HOSu), 6-Chloro-1-hydroxy-benzotriazole(Cl-HOBt), N-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt),1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxy-benzotriazole (HOBt) and

ethyl 2-cyano-2-hydroxyimino

acetate (CHA).

CHA is available under trade name OXYMAPURE®. CHA has proved to be aneffective scavenger as racemization is more suppressed compared tobenzotriazole-based scavengers. In addition, CHA is less explosive thane.g. HOBt or Cl-HOBt, so that its handling is advantageous, and, as afurther advantage, the coupling progress can be visually monitored by acolour change of the reaction mixture.

Preferably, HOBt or CHA, more preferably HOBt is used.

In a preferred embodiment, the combination of reagents in the HSPPSreaction is selected from the group consisting of TCTU/Cl-HOBt/DIPEA,TOTU/CHA/DIPEA and PyBop/HOBt/DIPEA.

As solvent, any inert liquid solvent, which can dissolve the reactants,may be used in HSPPS.

Preferred solvents are selected from the group consisting of dimethylsulfoxide (DMSO), dioxane, tetrahydrofuran (THF), 1-methyl-2-pyrrolidone(NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA),pyridine, dichloromethane (DCM), dichloroethane (DCE), chloroform,dioxane, tetrahydropyran, ethyl acetate, toluene, acetonitrile andmixtures thereof.

More preferred solvents are NMP, DMF and mixtures thereof.

Preferably, HSPPS is done at a temperature of from 0 to 50° C., morepreferably of from 5 to 30° C., even more preferably of from 15 to 25°C.

Preferably, HSPPS is done at atmospheric pressure.

Preferably, the reaction time for HSPPS is of from 15 min to 20 h, morepreferably of from 30 min to 5 h, even more preferably of from 30 min to2 h.

The term “part” in this description of reaction conditions of HSPPS ismeant to be a factor of the parts by weight of the combined peptidematerial, if not otherwise stated.

Preferably, of from 1 to 30 parts, more preferably of from 5 to 10parts, of solvent are used.

Preferably, of from 0.9 to 5 mol equivalents, more preferably of from 1to 1.5 mol equivalents, of coupling reagent is used, the mol equivalentbeing based on the mol of reactive C-terminal carboxy groups.

Preferably, of from 0.1 to 5 mol equivalents, more preferably of from0.5 to 1.5 mol equivalents, of coupling additive is used, the molequivalent being based on the mol of coupling reagent.

Preferably, of from 1 to 10 mol equivalents, more preferably of from 2to 3 mol equivalents, of tertiary base is used, the mol equivalent beingbased on the mol of coupling reagent.

If the N-terminally and C-terminally protected PEP, which was preparedaccording to method(A), represents the target peptide, preferably theN-terminal protecting group and the C-terminal protecting group and anyside chain protecting group are removed after the preparation accordingto method(A), to provide for the unprotected peptide PEP. This isusually called global deprotection.

The global deprotection conditions, which need to be applied, depend onthe nature of the chosen PG. Preferably, the involved PGs are selectedto allow global deprotection under weak, strong or reductive cleavingconditions, as defined above, depending on the nature of PGs.

The C-terminal protecting group of PEP, i.e. the DKP-PG, can be cleavedby conditions applicable for cleaving the respective handle group HGfrom the peptidyl radical, these conditions are known in peptidechemistry. Usually, the conditions are either reductive, weak or strongcleaving conditions, as defined above.

Preferably, the handle group HG is chosen to be cleavable under acidicconditions from the peptidyl radical PEP, and in this case, if theN-terminal protecting group of fragment PEP-N is a basic cleavable typeprotecting group or a reductively cleavable type protecting group, theN-terminal protecting group and the C-terminal protecting group and anyside chain protecting group are removed preferably after the preparationaccording to method(A) in two steps; but if the N-terminal protectinggroup of fragment PEP-N is acid type removable protecting group, theN-terminal protecting group and the C-terminal protecting group and anyside chain protecting group are removed preferably after the preparationaccording to method(A) in one step.

Any side chain protecting groups are typically retained until the end ofthe HSPPS. This deprotection reaction can be carried out underconditions applicable for the various side chain protecting groups,which have been used, and these conditions are known in peptidechemistry. In the case that different types of side chain protectinggroups are chosen, they may be cleaved successively. Advantageously, theside chain protecting groups are chosen, so that they are cleavablesimultaneously, and more advantageously concomitantly with N-terminalprotecting group of PEP.

Usually, side chain PGs are cleaved by strong, weak or reductivecleaving conditions as defined above.

Further subject of the invention is the use (A) of compound of formula(III-H), with the compound of formula (III-H) being as defined above,also with all its preferred embodiments, for the preparation of apeptide PEP;

preferably the use (A) of compound of formula (III-H), with the compoundof formula (III-H) being as defined above, also with all its preferredembodiments, in homogeneous solution phase peptide synthesis for thepreparation of a peptide PEP by a coupling reaction of the compound offormula (III-H) with an N-terminally protected amino acid or with anN-terminally protected PEP-N, with PEP-N being as defined above, alsowith all its preferred embodiments.

Use (A) is an embodiment of the above defined use of C-PEP in HSPPS.

Further subject of the invention is a method(B) for the preparation of acompound of formula (III-H), with the compound of formula (III-H) beingas defined above, also with all its preferred embodiments, characterizedby cleaving a protecting group PGXaaC^((pc)) from a compound of formula(III-PGXaaC^((pc)));

wherein

-   PGXaaC is an N-terminal protecting group conventionally used in SPPS    and is selected from the group consisting of basic cleavable type    protecting groups, acid cleavable type protecting groups and    reductively cleavable type protecting groups;-   pc, XaaC, PEP-C, HG, n, SG, Xaa1 and Xaa2 have the same definition    as above, also with all their preferred embodiments,-   in PGXaaC^((pc)), the index (pc) defines PGXaaC^((pc)) as the    N-terminal protecting group of PEP-C;    with the proviso, that PGXaaC^((pc)) is chosen to be of such a    cleavable type protecting group, that PGXaaC^((pc)) can be cleaved    from PEP-C without cleaving PEP-C from HG.

Preferably, if PEP-C carries side chain PGs, PGXaaC^((pc)) is chosen tobe of such a cleavable type protecting group, that PGXaaC^((pc)) can becleaved from PEP-C without cleaving any side chain PGs from PEP-C.

-   PGXaaC^((pc)) therefore is the protecting group of the N-terminal    amino acid residue of PEP-C, which is an embodiment of N-PG.

Method(B) is comprised in above defined method(C-PEP).

Compound of formula (III-PGXaaC^((pc))) is an embodiment of the abovedefined C-PEP.

If PGXaaC^((pc)) is a basic type PG, it is preferably Fmoc.

If PGXaaC^((pc)) is a strong type PG, it is preferably Boc.

If PGXaaC^((pc)) is a weak type PG, it is preferably Trt.

If PGXaaC^((pc)) is a reductive type PG, it is preferably Alloc.

PGXaaC^((pc)) is, depending on its type, cleaved by strong, weak, basicor reductive cleaving conditions, these conditions being as definedabove.

Further subject of the invention is a method(C) for the preparation of acompound of formula (III-PGXaaC^((pc))), with the compound of formula(III-PGXaaC^((pc))) being as defined above, also with all its preferredembodiments, method(C) comprises the consecutive steps a) and b),wherein

in step a) a protecting group PG2 is cleaved from a compound of formula(II-PG2)

wherein in formula (II-PG2)

-   PGXaaC^((pc)), PGXaaC, pc, PEP-C, HG, n, SG, Xaa1 and Xaa2 have the    same definition as above, also with all their preferred embodiments;-   PG2 is an N-terminal protecting group conventionally used in peptide    chemistry and is selected from the group consisting of basic    cleavable type protecting groups, acid cleavable type protecting    groups and reductively cleavable type protecting groups;    the alpha amino group of Xaa2 is protected by PG2,    ResinA being a resin used conventionally as solid phase in SPPS;    the 1-carboxy group of Xaa1 is coupled to a functional group of    ResinA;    to provide the compound of formula (II-H);

wherein in formula (II-H)

-   PGXaaC^((pc)), PGXaaC, pc, PEP-C, HG, n, SG, Xaa1, Xaa2 and ResinA    have the same definition as above, also with all their preferred    embodiments;-   the hydrogen H denoted with (2) is a hydrogen of the unprotected    alpha amino group of the amino acid residue Xaa2;    with the proviso, that PG2 is chosen to be of such a cleavable type    protecting group, that PG2 can be cleaved from Xaa2 without cleaving    PEP-C from HG;    and    in step b) the ResinA is cleaved from Xaa1 by an intra molecular    ring formation reaction reaction(INRIFO) between the alpha amino    group of Xaa2 and the carboxylic group of Xaa1 of compound of    formula (II-H), reaction(INRIFO) forms a cyclic dipeptide of Xaa1    and Xaa2, to provide the compound of formula (III-PGXaaC^((pc));    with the proviso, that the connection between ResinA and Xaa1 is    chosen to be cleavable under such cleaving condition, that ResinA    can be cleaved from Xaa1 by said reaction(INRIFO) without cleaving    PEP-C from HG.

This means, that PG2 is chosen to be of such a cleavable type protectinggroup and HG is chosen to be cleavable under such cleaving conditiondifferent from those cleaving conditions needed to cleave PG2 from Xaa2,that PG2 can be cleaved from Xaa2 without cleaving PEP-C from HG;

and this means also,that the connection between ResinA and Xaa1 is chosen, i.e. ResinA ischosen, to be cleavable under such cleaving condition, that ResinA canbe cleaved from Xaa1 by reaction(INRIFO) without cleaving PEP-C from HG.

Preferably, if PEP-C carries side chain PGs, PG2 is chosen to be of sucha cleavable type protecting group, that PG2 can be cleaved from Xaa2without cleaving any side chain PGs from PEP-C.

Preferably, if PEP-C carries side chain PGs, the connection betweenResinA and Xaa1 is chosen to be of such a cleavable type protectinggroup, that ResinA can be cleaved from Xaa1 by reaction(INRIFO) withoutcleaving any side chain PGs from PEP-C.

Compound of formula (II-PG2) is an embodiment of the above definedPEP-C-DKP-L-ResinA.

-   The dipeptide in e.g. formula (II-H) is one embodiment of the above    mentioned DPR, which forms the DKP, i.e. the diketopiperazine    residue.    PGXaaC^((pc)) and PG2 can be different protecting groups, which are    cleaved under different reaction conditions; in this case,    deprotection of the N-terminus of PEP-C and deprotection of Xaa2,    i.e. method(B) and method(C) are done consecutively.

But preferably, PGXaaC^((pc)) and PG2 are identical or at least are suchdifferent protecting groups, which are cleavable under the same reactionconditions; in this case, deprotection of the N-terminus of PEP-C anddeprotection of Xaa2, i.e. cleavage of PGXaaC^((pc)) from the compoundof formula (III-PGXaaC^((pc)), which is method(B), and cleavage of PG2from the compound of formula (II-PG2), which is step (a) of method(C),can be done simultaneously in one step.

PG2 is, depending on its type, cleaved by strong, weak, basic orreductive cleaving conditions, these conditions being as defined above.

-   Preferably, PG2 is selected from the group consisting of Fmoc,    Alloc, Boc, Trt, Mtt, Mmt and Ddz.    If PG2 is a strong type PG it is preferably Boc.    If PG2 is a weak type PG it is preferably Trt.    If PG2 is a reductive type PG it is preferably Alloc.    If PG2 is a basic type PG it is preferably Fmoc.-   ResinA is a resin conventionally used as solid phase in SPPS and the    bond between ResinA and Xaa1 can be cleaved under conditions, which    do not cleave an amide bond between two amino acid residues of a    peptide.-   Preferably, ResinA is a resin with functional groups, which is    conventionally used as solid support in SPPS, the functional groups    being NH₂ or OH.-   Preferably, ResinA is coupled to the 1-carboxylic acid group of Xaa1    by an ester or amide bond.-   More preferably, ResinA is chosen to be such a resin, that ResinA is    coupled to the C1-atom of the carboxy group of Xaa1 by an ester or    amide bond, neither of the bonds being cleavable under basic, weak    or reductive cleaving conditions.-   Preferably, ResinA is selected from the group consisting of    hydroxymethylpolystyrene (HMPS) resins, polyethylenglycol (PEG)    based resins, resins, wherein PEG is grafted on a resin different    from a PEG resin, polystyrene resin, p-benzyloxybenzyl alcohol    resins, chloromethyl polystyrene-divinylbenzene resins, poly(vinyl    alcohol)-graft-poly(ethylene glycol) (PVA-g-PEG) resins.-   Resins, wherein PEG is grafted on a resin different from a PEG    resin, are preferably PEG grafted on polystyrene resin, on    p-benzyloxybenzyl alcohol resin or on chloromethyl    polystyrene-divinylbenzene resin.-   More preferably, ResinA is a HMPS resin or a chloromethyl    polystyrene-divinylbenzene resin.-   HydroxyChemMatrix® resins have a ChemMatrix® support, which is a    polyethylene glycol (PEG) support, and are an example for    polyethylene glycol based resins.-   HydroxyTentagel® resins have a Tentagel® support, which is a grafted    copolymer consisting of a low cross-linked polystyrene matrix on    which polyethylene glycol (PEG) is grafted, and are an example for    polystyrene based resins.-   p-Benzyloxybenzyl alcohol resins are called Wang resins.-   Chloromethyl polystyrene-divinylbenzene resin are called Merrifield    resins.

Step (a) and step (b) may require different reaction condition, i.e.step (a) and step (b) can be done consecutively.

Preferably, step (a) and step (b) require the same reaction conditions,i.e. step (a) and step (b) are done simultaneously in one step.

Preferably, method(B), i.e. cleavage of PGXaaC^((pc)) from the compoundof formula (III-PGXaaC^((pc)), step (a) of method(C), i.e. cleavage ofPG2 from the compound of formula (II-PG2), and step (b) of method(C),i.e. the reaction(INRIFO), require the same reaction conditions andtherefore can be done simultaneously in one step.

Preferably, step (b), the reaction(INRIFO), which cleaves Xaa1 from theResinA, is done in a solvent (b).

Step (b) preferably is done at conditions, which afford for the alphaamino group of Xaa2 to be in a deprotonated state as an unprotonatedamino group, i.e. not to be present as an ammonium ion.

More preferably, step (b) is done by the addition of at least one base(b).

If step (a) was done in acidic conditions, the pH is preferablyneutralised by the addition of a base, preferably a tertiary base, morepreferably the tertiary base is one of those used in HSPPS as mentionedabove. To induce the reaction(INRIFO) of step (b), a base (b) is added,preferably the base (b) is a secondary amine, more preferably theconjugated acid of said secondary amine has a pKa value of from 5.0 to15, more preferably of from 7.5 to 10. Said secondary amine ispreferably a dialkylamine, more preferably it is selected from the groupconsisting of dimethylamine, di-n-propylamine, diethylamine,alpha-(p-tolyl)pyrroline, pyrrolidine, alpha-ethylpyrrolidine,alpha-benzylpyrrolidine, alpha-cyclohexylpyrrolidine, morpholine,piperidine, 2-methylpiperidine, N,N-dimethylhydroxylamine and N—C₁₋₄alkylanilines, with the C₁₋₄ alkyl in the N—C₁₋₄ alkylanilines beinglinear or branched and selected from the group consisting of methyl,ethyl, n-propyl, iso-propyl, n-butyl and isobutyl, more preferably saidC₁₋₄ alkyl being ethyl.

As solvent (b), any inert solvent, which can dissolve the reactants, maybe used. Preferably, solvent (b) is selected from the group consistingof dimethyl sulfoxide (DMSO), dioxane, tetrahydrofuran (THF),1-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF),N,N-dimethylacetamide (DMA), pyridine, dichloromethane (DCM),dichloroethane (DCE), chloroform, dioxane, tetrahydropyran, ethylacetate, toluene, acetonitrile and mixtures thereof.

More preferably, solvent (b) is selected from the group consisting ofNMP, DMF, THF and mixtures thereof.

Preferably, step (b) is done at a temperature of from 0 to 50° C., morepreferably of from 5 to 30° C., even more preferably of from 15 to 25°C.

Preferably, step (b) is done at atmospheric pressure.

Preferably, the reaction time for step (b) is of from 1 min to 1 h, morepreferably of from 1 min to 30 min, even more preferably of from 5 minto 15 min.

The term “part” in this description of step (b) is meant to be a factorof the parts by weight of the treated material, which is the compound offormula (II-H), if not otherwise stated.

Preferably, of from 5 to 20 parts, more preferably of from 5 to 15 partsof solvent are used.

Preferably, the amount of base (b) is of from 30 to 1% by weight, morepreferably of from 15 to 2% by weight, even more preferably of from 10to 5% by weight, with the % by weight being based on the total weight ofthe compound of formula (II-H).

Further subject of the invention is a method(D) for the preparation of acompound of formula (II-PG2), with the compound of formula (II-PG2)being as defined above, also with all its preferred embodiments,

characterized by the sequential addition of the amino acids of PEP-C ofcompound of formula (II-PG2), except for the C-terminal amino acid ofPEP-C, to a compound of formula (II-XaaC⁽¹⁾) by conventional solid phasepeptide synthesis SPPS methodology, comprising the necessary andconventional steps of repetitive SPPS cycles such as deprotecting theN-terminus of the C-terminal amino acid attached to the resin, couplingthe next amino acid, deprotecting, if more amino acids have to becoupled, the N-terminus of the thus coupled amino acid and so on,

-   starting the SPPS with deprotecting the N-terminus of Xaa⁽¹⁾ and    coupling of the amino acid of the second position from the    C-terminus of PEP-C, said amino acid of the second position from the    C-terminus of PEP-C having the formula PGXaaC⁽²⁾-XaaC⁽²⁾-OH; and,-   continuing the SPPS, in case that pc is 3 or greater, consecutively    with any next amino acid of formula    PGXaaC^((iippcc))-XaaC^((iippcc))-OH according to the sequence of    PEP-C, with iippcc being an integer of from 3 to (pc-1); and-   ending the SPPS with the addition of the N-terminal amino acid of    PEP-C, said N-terminal amino acid having the formula    PGXaaC^((pc))-XaaC^((pc))-OH;

wherein

-   PGXaaC^((pc)), PGXaaC, XaaC, pc, PEP-C, HG, n, SG, PG2, Xaa1, Xaa2    and ResinA have the same definition as above, also with all their    preferred embodiments;-   in PGXaaC⁽¹⁾, PGXaaC⁽²⁾ and PGXaaC^((iippcc)), the indices (1), (2)    and (iippcc) define the respective PGXaaC as the protecting group of    the amino group of the respective amino acid residue XaaC of PEP-C;-   in XaaC⁽¹⁾, XaaC⁽²⁾ and XaaC^((iippcc)), the indices (1), (2) and    (iippcc) define the respective XaaC as the respective amino acid    residue XaaC of PEP-C;-   with the proviso, that PGXaaC⁽¹⁾, PGXaaC⁽²⁾ and any    PGXaaC^((iippcc)) are protecting groups different from PG2 and that    they are cleavable under reaction conditions different from those    needed to cleave PG2 from Xaa2,-   and with the further proviso, that the reaction conditions used to    cleave PGXaaC⁽¹⁾, the reaction conditions used to cleave the    N-terminal protecting group PGXaaC⁽²⁾ of said second amino acid and    the reaction conditions used to cleave any N-terminal protecting    group PGXaaC^((iippcc)) of said next amino acids, do not cleave PG2    from Xaa2;-   and with the further proviso, that the bond between Xaa1 and ResinA    is of such a type, that is it not cleaved during the SPPS.

Preferably, if any XaaC carries side chain PGs, any PGXaaC is chosen tobe of such a cleavable type protecting group, that any PGXaaC can becleaved from its amino acid XaaC without cleaving any side chain PGsfrom any side chain protected XaaC.

-   Therefore, PGXaaC⁽¹⁾ and PGXaaC⁽²⁾ are identical or different and    independently from each other N-terminal protecting groups    conventionally used in SPPS and are the N-terminal protecting groups    of the amino acids XaaC⁽¹⁾ and XaaC⁽²⁾ of PEP-C respectively, to    which the next amino acid of PEP-C is added by the SPPS, and are    selected from the group consisting of basic cleavable type    protecting groups, acid cleavable type protecting groups and    reductively cleavable type protecting groups.-   In the case, that pc is 2, then PEP-C is a dipeptidyl radical, and    said amino acid of the second position from the C-terminus of PEP-C,    having the formula PGXaaC⁽²⁾-XaaC⁽²⁾-OH, is identical with    PGXaaC^((pc))-XaaC^((pc))-OH, and no said amino acid    PGXaaC^((iippcc))-XaaC^((iippcc))-OH is used.

In stead of using only individual amino acids as building blocks, alsooligopeptides, preferably di- or tripeptides, more preferablydipeptides, can be used as building blocks in SPPS. This is e.g. known,when pseudoproline is used as a side chain protecting group, in thiscase conventionally the respective dipeptide is used in SPPS as buildingblock.

Preferably, PG2 is Fmoc or Alloc, and

-   -   PGXaaC⁽¹⁾, PGXaaC⁽²⁾ and any PGXaaC^((iippcc)) are Boc.

-   In another preferred embodiment, PG2 is selected from the group    consisting of Boc, Trt, Mtt, Mmt, Ddz and Alloc, and    -   PGXaaC⁽¹⁾, PGXaaC⁽²⁾ and any PGXaaC^((iippcc)) are Fmoc.        More preferably, PG2 is Trt, Boc or Alloc, and    -   PGXaaC⁽¹⁾, PGXaaC⁽²⁾ and any PGXaaC^((iippcc)) are Fmoc.        Even more preferably, PG2 is Trt or Alloc, and    -   PGXaaC⁽¹⁾, PGXaaC⁽²⁾ and any PGXaaC^((iippcc)) are Fmoc.

Typical reaction conditions and parameters and reagents and standardprotocols for SPPS are known in the art, e.g. Lloyd-Williams et al.,“Chemical Approaches to the Synthesis of Peptides and Proteins”, CRCPress, 1997, or Chan et al., “Fmoc solid phase peptide synthesis”,Oxford University Press, 2000.

SPPS may be carried out by standard methods known in peptide chemistry.Usually, in SPPS the amino acids are added from the C-terminus to theN-terminus. Thus, the C-terminal amino acid or the peptide groupproximal to the C terminus of a desired peptide fragment is the first tobe added to the solid support, the resin. This occurs by reacting thecarboxy group of the C-terminus to a complementary functional group onthe resin support, the N-terminal amino group being usually protected bya protecting group in order to prevent undesired side reactions. In casethat any amino acid or peptide group to be added has reactive groups onside chains, they are protected by protecting groups as well in order toavoid undesired side reactions. After coupling the first amino acid orpeptide fragment to the solid support, the N-terminal protecting groupis removed, and the next, N-terminally protected amino acid or peptidefragment is coupled to the first one. Then in successive cycles ofremoval of the N-terminal protecting group and coupling, the amino acidsor peptide groups are consecutively attached to previously elongatedpeptidyl radical until the desired peptidyl radical is formed.

The product of solid phase synthesis is thus a peptidyl radical bound toa solid support.

A wide variety of solid supports for SPPS are known. Preferably, thesolid support comprises a resin that is made from one or more polymers,copolymers or combinations of polymers such as polyamide, polysulfamide,substituted polyethylenes, polyethyleneglycol, phenolic resins,polysaccharides, or polystyrene.

The solid support should be sufficiently insoluble and inert to solventsused in peptide synthesis.

The solid support typically includes a linking moiety having thefunctional group, to which the first amino acid or first peptide isinitially coupled. The peptidyl radical is cleaved from the solidsupport under the appropriate reaction conditions to release the peptidefrom the support. Suitable solid supports can have linkers that arephoto-cleavable, acid cleavable, preferably by TFA or HF, fluoride ioncleavable, reductively cleavable, preferably by Pd(0) catalysis;nucleophilically cleavable or cleavable by radicals. Preferably, thelinking moiety of the solid support is chosen, that either the peptidylradical is cleavable under such conditions that the side chainprotecting groups of the peptide are not removed, or that the peptidylradical is cleavable under such conditions that the side chainprotecting groups of the peptide are simultaneously and completelyremoved as well.

Preferably, SPPS is done with an acid cleavable solid support, morepreferably the linking moiety of the solid support comprises tritylgroups, such as chlorinated trityl resins, preferably 2-chlorotritylchloride (2-CTC) resin, or 4-methyltrityl chloride resins,4-methoxytrityl chloride resins, 4-aminobutanl-ol 2-chlorotrityl resins,4-aminomethylbenzoyl-2-chlorotrityl resins, 3-aminopropan-1-ol2-chlorotrityl resins, bromoacetic acid 2-chlorotrityl resins,cyanoacetic acid 2-chlorotrityl resins, 4-cyanobenzoic acid2-chlorotrityl resins, glicinol-2-chlorotrityl resins, propionic2-chlorotrityl resins, ethyleneglycol-2-chlorotrityl resins, N-Fmochydroxylamine 2-chlorotrityl resins or hydrazine 2-chlorotrityl resins.Other preferred solid supports are polystyrene resins, or resins basedon copolymers of styrene and divinylbenzene, having functional groups tobond the C-terminal carboxy group, preferably Wang resins, whichcomprise a copolymer of styrene and divinylbenzene with4-hydroxymethylphenyloxymethyl anchoring groups, further resins such as4-hydroxymethyl-3-methoxyphenoxybutyric acid resin.

Preferred resins are Wang, (2-CTC) and 4-hydroxymethyl-3-methoxyphenoxybutyric acid resins.

In order to prepare a resin for solid phase synthesis, the resin can bepre-washed with one or more suitable solvents.

As solvents, which are preferably used in SPPS, the preferred solventsmentioned above under HSPPS may also be used in SPPS. More preferredsolvents are NMP, DMF, DCM mixtures thereof.

More preferred mixtures are DMF:DCM with a volume ratio of from 9:1 to1:9, more preferred of from 4:1 to 1:4.

The SPPS preferably is done with any side chain of amino acids, whichhas a reactive functional group, being protected by side chainprotecting groups in order to avoid undesired side reactions. The natureand use of side chain protecting groups is well known in the art.

The choice of a side chain-protecting group can depend on variousfactors, for example, type of synthesis performed, processing to whichthe peptide will be subjected, and the desired intermediate product orfinal product. The nature of the side chain protecting group alsodepends on the nature of the amino acid itself. Generally, a side chainprotecting group is chosen that is not removed during deprotection ofthe alpha-amino groups during the solid phase synthesis. Therefore theprotecting group of the alpha amino group and any side chain protectinggroup are typically not the same, preferably they represent anorthogonal system. The term “orthogonal system” is defined in Baranay,G., and Merrifield, R. B., JACS, 1977, 99, 22, 7363-7365.

Examples of side chain protecting groups include acetyl (Ac), benzoyl(Bz), tert-butyl (tBu), triphenylmethyl (Trt), tetrahydropyranyl, benzylether (Bzl), 2,6-dichlorobenzyl ether (DCB), tert-butoxycarbonyl (Boc),4-nitrobenzenesulfonyl (Ns), p-toluenesulfonyl (Tos),pentamethyldihydrobenzohran-5-sulfonyl (Pbf),1,2-dimethyl-indole-3-sulfonyl (MIS), adamantyloxycarbonyl, xanthyl(Xan), methyl ester, ethyl ester, tert-butyl ester (OtBu),benzyloxycarbonyl (Z), 2-chlorobenzyloxycarbonyl(2-Cl-Z),tert-amyloxycarbonyl (Aoc), aromatic or aliphatic urethane typeprotecting groups, photo labile groups such as nitro veratryloxycarbonyl(NVOC); and fluoride labile groups such as trimethylsilyloxycarbonyl(TEOC).

Preferred side chain groups are tBu, Trt, Boc, Tos, Pbf, OtBu and Z.

Preferably, functional groups containing amino acids commonly used withside chain protecting groups are Arg(Pbf), Asp(OtBu), Gln(Trt),Glu(OtBu), His(Trt), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc) andTyr(tBu), with Arg also sometimes used without side chain protectinggroup.

E.g. Fmoc-Arg(Pbf)-OH has formula (ARG-PBF).

An N-terminal protecting group is removed in a deprotection reactionprior to the addition of the next amino acid to be added to the growingpeptide chain, but can be maintained when the peptide is cleaved fromthe support. The choice of an N-terminal protecting group can depend onvarious factors, for example, type of synthesis performed and thedesired intermediate product or final product.

Examples of amino-terminal protecting groups include

-   (1) acyl-type protecting groups, such as formyl, acrylyl (Acr),    benzoyl (Bz) and acetyl (Ac);-   (2) aromatic urethane-type protecting groups, such as    benzyloxycarbonyl Z and substituted Z, such as    p-chlorobenzyloxycarbonyl, p nitrobenzyloxycarbonyl,    p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl;-   (3) aliphatic protecting groups such as t-butyloxycarbonyl (Boc),    2-phenylpropyl (2)-oxycarbonyl (Poc), 2-(4-biphenylyl)-propyl (2)    oxycarbonyl (Bpoc), diisopropylmethoxycarbonyl,    isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl (Alloc);-   (4) cycloalkyl urethan-type protecting groups, such as    9-fluorenyl-methyloxycarbonyl (Fmoc), cyclopentyloxycarbonyl,    adamantyloxycarbonyl, and cyclohexyloxycarbony 1;-   (5) thiourethantype protecting groups, such as phenylthiocarbonyl.

Preferred N-terminal protecting groups are Fmoc, Bpoc, Poc and Boc.

Fmoc or Fmoc-like chemistry is highly preferred for solid phase peptidesynthesis, inasmuch as cleaving the resultant peptide in a protectedstate from the resin is relatively straightforward to carry out usingmildly acidic cleaving agents. This kind of cleaving reaction isrelatively clean in terms of resultant by-products, impurities, etc.Furthermore, the Fmoc protecting group of the N-terminus fits well withthe above mentioned side chain protecting groups in order to representan orthogonal system.

Coupling in SPPS is usually done with a coupling reagent, preferably inthe presence of a tertiary base, further preferably in the presence of acoupling additive, and further preferably the coupling reagent and anyother compound is dissolved in a SPPS solvent as mentioned above.

The reaction conditions and reaction reagents for SPPS and for HSPPS areoften similar.

Typical coupling reagents used in SPPS are phosphonium and uroniumsalts, mixed anhydrides, carbodiimides, other acylating agents such asactivated esters or acid halogenides, and activatedbenzotriazinderivatives.

Phosphonium and uronium salts are preferably those used in HSPPS asmentioned above.

A mixed anhydride is for instance propane phosphonic acid anhydride(T3P).

Carbodiimide coupling reagents are preferably those used in HSPPS asmentioned above.

An activated esters is for instance isobutyl-chloroformiate (ICBF).

An activated benzotriazinderivatives is for instance3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT).

The tertiary base is preferably one of those used in HSPPS as mentionedabove.

Coupling additives used in SPPS are those used in HSPPS as mentionedabove.

The amount of the second and of the subsequent amino acids used isusually of from 1 to 3 mol equivalents relative to the loading factorachieved by the first coupling reaction on the resin support, preferablyof from 1.3 to 3 mol equivalents, more preferably of from 1.5 to 3 molequivalents.

Preferably, SPPS is done at a temperature of from 0 to 50° C., morepreferably of from 5 to 30° C., even more preferably of from 15 to 25°C.

Preferably, SPPS is done at atmospheric pressure.

Preferably, the reaction time for SPPS is of from 15 min to 20 h, morepreferably of from 30 min to 5 h, even more preferably of from 30 min to2 h.

The term “part” in this description of reaction conditions of SPPS ismeant to be a factor of the parts by weight of the solid supportmaterial, if not otherwise stated.

Preferably, of from 1 to 30 parts, more preferably of from 5 to 10parts, of solvent are used.

Preferably, of from 0.9 to 5 mol equivalents, more preferably of from 1tol.5 mol equivalents, of coupling reagent is used, the mol equivalentbeing based on the mol of reactive carboxy groups, in case of SPPS ofreactive C-terminal carboxy groups.

Preferably, of from 0.1 to 5 mol equivalents, more preferably of from0.5 to 1.5 mol equivalents, of coupling additive is used, the molequivalent being based on the mol of coupling reagent.

Preferably, of from 1 to 10 mol equivalents, more preferably of from 2to 3 mol equivalents, of tertiary base is used, the mol equivalent beingbased on the mol of coupling reagent.

These SPPS conditions are general condition, which are applicable tocoupling a carboxy group comprising building block to an amino groupcomprising reaction partner. In case of SPPS, the carboxy groupcomprising building block is the N-terminally protected amino acid whichis to be coupled, and the amino group comprising reaction partner is theC-terminal amino acid or the growing peptide chain, which are connectedto the support material.

This means, that the amount of carboxy group comprising building blockused is usually of from 1 to 3 mol equivalents relative to mol of theamino group comprising reaction partner, preferably of from 1.3 to 3 molequivalents, more preferably of from 1.5 to 3 mol equivalents.

Further subject of the invention is a method(E) for the preparation of acompound of formula (II-XaaC⁽¹⁾), with the compound of formula(II-XaaC⁽¹⁾) being as defined above, also with all its preferredembodiments,

characterized by a coupling of an amino acid PGXaaC⁽¹⁾-XaaC⁽¹⁾-OH to acompound of formula (I-HG);

whereinthe hydrogen denoted with (3) is a hydrogen of an unprotected functionalgroup of HG;PGXaa⁽¹⁾, XaaC⁽¹⁾, PGXaaC, XaaC, HG, n, SG, PG2, Xaa1, Xaa2 and ResinAhave the same definition as above, also with all their preferredembodiments.

In case of formula (I-HG), the (*) in the any of the above definitionsof HG now denotes in formula (I-HG) the bond between the hydrogendenoted with (3) and HG.

Method(E) is analogous to above defined step (i).

-   In case, that HG is a handle group selected from the group    consisting of handle group of formula (HGF-I) in case that T1-1 is    NH, handle group of formula (HGF-II), handle group of formula    (HGF-III) and handle group of formula (HGF-VI),    the hydrogen denoted with (3) is connected to the terminal nitrogen    of HG, and reaction conditions and parameters and reagents and    standard protocols for the method(E) are preferably those which have    been described above for the SPPS;    with the carboxy group comprising building block being the amino    acid PGXaaC⁽¹⁾-XaaC⁽¹⁾-OH, and the amino group comprising reaction    partner being the compound of formula (I-HG), which are connected to    the support material.-   In case, that HG is a handle group selected from the group    consisting of handle group of formula (HGF-I) in case that T1-1 is    O, handle group of formula (HGF-IV) and handle group of formula    (HGF-V),    the hydrogen denotes with (3) is connected to the terminal oxygen of    HG, and preferably, the coupling according to method(E) is done    according to a method(E-OH) using in a solvent (E-OH), using one or    more coupling reagents (E-OH), and is done preferably in the    presence of one or more coupling additives (E-OH).

Preferable coupling reagents (E-OH) are carbodiimide coupling reagents(E-OH).

Carbodiimide coupling reagents (E-OH) are preferably selected from thegroup consisting of diisopropyl-carbodiimide (DIC),dicyclohexyl-carbodiimide (DCC) and water-soluble carbodiimides (WSCDI)such as 1-ethyl-3-(3-dimethylaminopropyl)

carbo

diimide (EDC)

Preferred coupling reagent (E-OH) is DIC.

A coupling additive (E-OH) is preferably DMAP or a nucleophilic hydroxycompound capable of forming activated esters, more preferably thenucleophilic hydroxy compound having an acidic, nucleophilic N-hydroxyfunction wherein N is imide or is N-acyl or N-aryl substituted triazeno,the triazeno type coupling additive being preferably aN-hydroxy-benzotriazol derivative (or 1-hydroxy-benzotriazol derivative)or a N-hydroxybenzotriazine derivative. Such coupling additives (E-OH)have been described in WO 94/07910 and EP 410 182. Since they also actas scavengers, they are also called scavengers.

Preferred coupling additives (E-OH) are selected from the groupconsisting of

DMAP, N-hydroxy-succinimide (HOSu), 6-Chloro-1-hydroxy-benzotriazole(Cl-HOBt), N-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt),1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxy-benzotriazole (HOBt) andethyl 2-cyano-2-hydroxyimino-acetate (CHA).CHA is available under tradename OXYMAPURE®. CHA has proved to be aneffective scavenger as racemization is more suppressed compared tobenzotriazole-based scavengers. In addition, CHA is less explosive thane.g. HOBt or Cl-HOBt, so that its handling is advantageous, and, as afurther advantage, the coupling progress can be visually monitored by acolour change of the reaction mixture.

Preferably, DMAP is used as coupling additive (E-OH).

As solvent, any inert liquid solvent (E-OH) may be used.

Preferred solvents (E-OH) are selected from the group consisting ofdimethyl sulfoxide (DMSO), dioxane, tetrahydrofuran (THF),1-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF),N,N-dimethylacetamide (DMA), pyridine, dichloromethane (DCM),dichloroethane (DCE), chloroform, dioxane, tetrahydropyran, ethylacetate, toluene, acetonitrile and mixtures thereof.

More preferred solvents (E-OH) are NMP, DMF, DCM and mixtures thereof.

Preferably, the coupling according to method(E-OH) is done at atemperature of from 0 to 50° C., more preferably of from 5 to 30° C.,even more preferably of from 15 to 25° C.

Preferably, the coupling according to method(E-OH) is done atatmospheric pressure.

Preferably, the reaction time for the coupling according to method(E-OH)is of from 15 min to 20 h, more preferably of from 30 min to 5 h, evenmore preferably of from 30 min to 2 h.

The term “part” in this description of reaction conditions of thecoupling according to method(E-OH) is meant to be a factor of the partsby weight of the combined solid support material, if not otherwisestated.

Preferably, of from 1 to 30 parts, more preferably of from 5 to 10parts, of solvent (E-OH) are used in the coupling according tomethod(E-OH).

Preferably, of from 0.1 to 5 mol equivalents, more preferably of from 1to 1.5 mol equivalents, of coupling reagent (E-OH) is used in thecoupling according to method(E-OH), the mol equivalent being based onthe mol of reactive groups of HG.

Preferably, of from 0.1 to 5 mol equivalents, more preferably of from0.5 to 1.5 mol equivalents, of coupling additive (E-OH) is used in thecoupling according to method(E-OH), the mol equivalent being based onthe mol of coupling reagent (E-OH).

In method(E) and method(E-OH) respectively, preferably of from 0.1 to 5mol equivalents, more preferably of from 1 to 1.5 mol equivalents, ofPGXaaC⁽¹⁾-XaaC⁽¹⁾-OH is used, the mol equivalent being based on the molof reactive groups of HG.

Reactive groups of HG, which remain unreacted after the coupling ofPGXaaC⁽¹⁾-XaaC⁽¹⁾-OH, are preferably capped, preferably the capping isdone with acetic anhydride.

These conditions for method(E-OH) are general condition, which areapplicable to coupling a carboxy group comprising building block to anOH or SH group comprising reaction partner. In case of method(E-OH), thecarboxy group comprising building block is the amino acidPGXaaC⁽¹⁾-XaaC⁽¹⁾-OH, and the OH or SH group comprising reaction partneris the compound of formula (I-HG).

Further subject of the invention is a method(F) for the preparation of acompound of formula (I-HG), with the compound of formula (I-HG) being asdefined above, also with all its preferred embodiments, method(F)comprises

-   a step (F1A) for the case that n is 0; or method(F) comprises-   a step (F3A) and a step (F3B) for the case that n is 1, with the    step (F3B) being done after the step (F3A); or method(F) comprises-   a step (F4);-   with n as defined above, also with all its preferred embodiments;    wherein    step (F3A) comprises a coupling reaction (F3A-Coup) of a compound of    formula (I-PG2), (4)

wherein

-   PG2, Xaa1, Xaa2 and ResinA have the same definition as above, also    with all their preferred embodiments;-   the H denoted with (4) is the hydrogen of FG, FG being as defined    above, also with all it preferred embodiments as defined above;-   with a compound SGroup;-   the compound SGroup is a conventional building block used in peptide    chemistry having two reactive functional groups SGroup-FunSiteN and    SGroup-FunSiteC, the reactive functional group SGroup-FunSiteN is    OH, SH or NH₂ and is used as a functionality resembling the alpha    amino group of an amino acid building block in peptide synthesis and    can be protected by a suitable protecting group PGSG, the reactive    functional group SGroup-FunSiteC is used as a functionality    resembling the carboxylic acid group of an amino acid building block    in peptide synthesis;-   PGSG is a protecting group conventionally used in peptide chemistry    for protecting the alpha amino group of an amino acid or the    N-terminus of a peptide, and is selected from the group consisting    of basic cleavable type protecting groups, acid cleavable type    protecting groups and reductively cleavable type protecting groups;    the compound SGroup is the precursor of SG, with SG being as defined    above, also with all its preferred embodiments;    in case, that the reactive functional group SGroup-FunSiteN of    compound SGroup is protected by a protecting group PGSG, than in a    consecutive step (F-ConC) after step (F3A) PGSG is cleaved from SG;-   with the proviso, that PGSG is different from PG2 and that PGSG is    cleavable under reaction conditions different from those needed to    cleave PG2 from Xaa2,-   and with the further proviso, that the reaction conditions used to    cleave PGSG do not cleave PG2 from Xaa2;-   and with the further proviso, that the reaction conditions used to    cleave PGSG in a step (F-ConC) do not cleave Xaa1 from ResinA;-   providing a compound of formula (I-SG);

wherein

-   SG, PG2, Xaa1, Xaa2 and ResinA have the same definition as above,    also with all their preferred embodiments;-   the H denoted with (5) is a hydrogen of the reactive functional    group SGroup-FunSiteN;    step (F3B) is a coupling reaction (F3B-Coup) of the compound of    formula (I-SG) with a compound HGroup;-   the compound HGroup is a conventional building block used in peptide    chemistry having two reactive functional groups HGroup-FunSiteN and    HGroup-FunSiteC, the reactive functional group HGroup-FunSiteN is    OH, SH or NH₂ and is used as a functionality resembling the alpha    amino group of an amino acid building block in peptide synthesis and    can be protected by a suitable protecting group PGHG, the reactive    functional group HGroup-FunSiteC is used as a functionality    resembling the carboxylic acid group of an amino acid building block    in peptide synthesis;-   PGHG is a protecting group conventionally used in peptide chemistry    for protecting the alpha amino group of an amino acid or the    N-terminus of a peptide, and is selected from the group consisting    of basic cleavable type protecting groups, acid cleavable type    protecting groups and reductively cleavable type protecting groups;    the compound HGroup is the precursor of HG, with HG being as defined    above, also with all its preferred embodiments;    step (F1A) comprises a coupling reaction (F1A-Coup) of the compound    of formula (I-PG2) with the compound HGroup, with the compound    HGroup being as defined above, also with all its preferred    embodiments;    in case, that the reactive functional group HGroup-FunSiteN of    compound HGroup is protected by a protecting group PGHG, than in a    consecutive step (F-ConA) after step (F3B) or after step (F1A), PGHG    is cleaved from HG;-   with the proviso, that PGHG is a protecting group different from PG2    and that PGHG is cleavable under reaction conditions different from    those needed to cleave PG2 from Xaa2,-   and with the further proviso, that the reaction conditions used to    cleave PGHG do not cleave PG2 from Xaa2;-   and with the further proviso, that the reaction conditions used to    cleave PGHG in a step (F-ConA) do not cleave Xaa1 from ResinA;-   the step (F4) comprises a coupling reaction of a compound of formula    (pDKP) with a ResinA,

wherein

-   HG, n, SG, PG2, Xaa1 and Xaa2 have the same definition as above,    also with all their preferred embodiments;-   with the ResinA having the same definition as above, also with all    its preferred embodiments;-   H-PGHG is hydrogen in case that the reactive functional group    HGroup-FunSiteN is OH or SH;-   H-PGHG is a protecting group PGHG in case that the reactive    functional group HGroup-FunSiteN is NH₂;-   with PGHG as defined above, also with all its preferred embodiments;-   in case, that H-PGHG is PGHG, than in a consecutive step (F-ConB)    after the reaction F4, PGHG is cleaved from HG;-   with the proviso, that PGHG is different from PG2 and that PGHG is    cleavable under reaction conditions different from those needed to    cleave PG2 from Xaa2,-   and with the further proviso, that the reaction conditions used to    cleave PGHG do not cleave PG2 from Xaa2;-   and with the further proviso, that the reaction conditions used to    cleave PGHG in step (F-ConB) do not cleave Xaa1 from ResinA.

The compound of formula (pDKP) ends C-terminally with the freecarboxylic acid group of Xaa1.

Compound HGroup is an embodiment of above mentioned HG used as buildingblock in method(DKP-L-ResinA) or in method(DKP-L).

Compound SGroup is an embodiment of above mentioned SG used as buildingblock in method(DKP-L-ResinA) or in method(DKP-L).

The compound of formula (DKP-L-ResinA-F) and the compound of formula(I-HG) are embodiments of above defined DKP-L-ResinA,

with H-PGHG, HG, SG, n PG2, Xaa2, Xaa1 and ResinA being as definedabove, also with all their preferred embodiments.

Compound of formula (DKP-L-ResinA-F) is the product of the reaction F1or F3, optionally with the consecutive step (F-ConA) or (F-ConC), or ofthe reaction F4 optionally with the consecutive step (F-ConB).

The compound of formula (pDKP) is an embodiment of above defined DKP-L.

Compound of formula (I-PG2) and compound of formula (I-SG) areembodiments of intermediates of above defined method(DKP-L-ResinA).

In case of step (F3A) or in case of step (F1A), and FG being NH₂, thecoupling reaction (F3A-Coup) or the coupling reaction (F1A-Coup) ispreferably done under reaction conditions and parameters and reagentsand protocols as described above for the SPPS, also with all thedescribed preferred embodiments, with the mentioned carboxy groupcomprising building block being the compound SGroup or the compoundHGroup respectively, and the amino group comprising reaction partnerbeing the compound of formula (I-PG2).

In case of step (F3A) or in case of step (F1A), and FG being OH or SH,the coupling reaction (F3A-Coup) or the coupling reaction (F1A-Coup) ispreferably done under the reaction conditions and parameters andreagents and protocols as described above for the method(E-OH), with thecarboxy group comprising building block being the compound SGroup or thecompound HGroup respectively, and the OH or SH group comprising reactionpartner being the compound of formula (I-PG2).

In case of step (F3B) and the reactive functional group SGroup-FunSiteNbeing NH₂, the coupling reaction (F3B-Coup) is preferably done underreaction conditions and parameters and reagents and protocols asdescribed above for the SPPS, also with all the described preferredembodiments, with the mentioned carboxy group comprising building blockbeing the compound HGroup, and the amino group comprising reactionpartner being the compound of formula (I-SG).

In case of step (F3B) and the reactive functional group SGroup-FunSiteNbeing OH or SH, the coupling reaction (F3B-Coup) is preferably doneunder the reaction conditions and parameters and reagents and protocolsas described above for the method(E-OH), with the carboxy groupcomprising building block being the compound HGroup, and the OH or SHgroup comprising reaction partner being the compound of formula (I-SG).

In the compound of formula (pDKP), the carboxylic acid group of Xaa1 isunprotected. This unprotected carboxylic acid group of Xaa1 is reactedwith an unprotected functional group of ResinA in the step (F4).Therefore, the coupling reaction in step (F4) is analogue to aconventional coupling reaction of an amino acid to a resin, i.e. thesolid support. The coupling of an amino acid to a solid support is aknown reaction, therefore the reaction conditions and parameters of thecoupling reaction in step (F4) are known.

Preferably, if the functional group of ResinA, which is to be coupled tothe carboxylic acid group of Xaa1 of compound of formula (pDKP), is aNH₂ group, the coupling reaction in step (F4) is preferably done underreaction conditions and parameters and reagents and protocols asdescribed above for the SPPS, also with all the described preferredembodiments, with the mentioned carboxy group comprising building blockbeing the compound of formula (pDKP), and the amino group comprisingreaction partner being the ResinA.

Preferably, if the functional group of ResinA, which is to be coupled tothe carboxylic acid group of Xaa1 of compound of formula (pDKP), is OHor SH, the coupling reaction in step (F4) is preferably done under thereaction conditions and parameters and reagents and protocols asdescribed above for the method(E-OH), with the mentioned carboxy groupcomprising building block being the compound of formula (pDKP), and theOH or SH group comprising reaction partner being ResinA.

-   Preferably, PG2 is Fmoc or Alloc, and    -   a possible PGHG or PGSG is Boc.-   In another preferred embodiment, PG2 is selected from the group    consisting of Boc, Trt, Mtt, Mmt, Ddz and Alloc, and    -   a possible PGHG or PGSG is Fmoc.        More preferably, PG2 is Trt, Boc or Alloc, and    -   a possible PGHG or PGSG is Fmoc.        Even more preferably, PG2 is Trt or Alloc, and    -   a possible PGHG or PGSG is Fmoc.-   Compound SGroup is the precursor of SG, with SG being as defined    above, also with all its preferred embodiments, and has two reactive    functional groups SGroup-FunSiteN and SGroup-FunSiteC, as explained    above in the context of method(DKP-L-ResinA). The reactive    functional group SGroup-FunSiteN preferably is OH or NH₂, more    preferably NH₂. The reactive functional group SGroup-FunSiteN is    present in the protected or coupled state as a connecting group    CG-SG, CG-SG being —O—, —S— or —N(H)—. The reactive functional group    SGroup-FunSiteC is can be unprotected or can be preactivated and is    the coupling site in the respective coupling reaction. Preferably,    the reactive functional group SGroup-FunSiteC is a carboxylic acid    group, if it is unprotected, or it is a preactivated carboxylic acid    group. After this coupling reaction, any protecting group of the    reactive functional group SGroup-FunSiteN is cleaved in order to    make the reactive functional group SGroup-FunSiteN available for the    next coupling reaction.    -   When the reactive functional group SGroup-FunSiteC a        preactivated carboxylic acid group, the preactivation is        preferably in a way as common in peptide chemistry. For example,        reactivated carboxylic acid groups are used in form of their        ester with N-hydroxysuccinimid or with pcnta flouro phenol.

Compound SGroup and SG respectively, can comprise ethylenoxid units of adefined and discrete number, or they can comprise a distribution ofethylene oxide units as is the case, when PEG chains are synthesized bypolymerization of ethylene oxide without subsequent separation of theindividual molecules of same chain length. In case of a distribution,compound SGroup, and thereby indirectly also SG, is specified rather byits average molecular weight and not by a discrete number of ethyleneoxide units. Preferably, molecular weights of compound SGroup are from1500 to 5000, preferably from 1500 to 4000, more preferably from 1500 to3500.

Preferably, PGSG is Alloc, Fmoc, Mmt or Z.

Preferably, compound SGroup is a compound SGroup1, SGroup2, SGroup3,SGroup4, SGroup5, SGroup 6, SGroup7 or SGroup8;

-   compound SGroup1 is the compound of formula (SG-I), wherein Fmoc is    connected via the bond denoted with (***) and OH is connected via    the bond denoted with (****) in formula (SG-I), preferably m1 is 3;-   compound SGroup2 is the compound of formula (SG-II), wherein Z, Fmoc    or Alloc is connected via the bond denoted with (***) and OH is    connected via the bond denoted with (****) in formula (SG-II),    preferably m5 is 2;-   compound SGroup3 is the compound of formula (SG-II), wherein Boc or    Fmoc is connected via the bond denoted with (***) and OH is    connected via the bond denoted with (****) in formula (SG-II),    preferably m5 is 1;-   compound SGroup4 is the compound of formula (SG-III), wherein Boc is    connected via the bond denoted with (***) and OH is connected via    the bond denoted with (****) in formula (SG-III), preferably m6 and    m7 are 2;-   compound SGroup5 is the compound of formula (SG-IV), wherein Mmt or    Boc is connected via the bond denoted with (***) and OH is connected    via the bond denoted with (****) in formula (SG-IV), preferably m9    is 4, 8, 12 or 27; especially the combination of Boc and m9 being 4,    8, 12 or 27; or the combination of Mmt and m9 being 4;-   compound SGroup6 is the compound of formula (SG-V), wherein Boc or    Fmoc is connected via the bond denoted with (***) and OH is    connected via the bond denoted with (****) in formula (SG-V),    preferably m10 is 1 or the molecular weight is 1500 to 3500, more    preferably 3000; especially the combination Fmoc with ml 0 being 1,    or the combination of Boc and the molecular weight being 1500 to    3500, more preferably 3000;-   compound SGroup7 is the compound of formula (SG-VI), wherein Boc is    connected via the bond denoted with (***) and Br is connected via    the bond denoted with (****) in formula (SG-VI), preferably ml 1 is    3;-   compound SGroup8 is the compound of formula (SG-VII), wherein Boc or    Fmoc is connected via the bond denoted with (***) and OH or    N-hydroxysuccinimid is connected via the bond denoted with (****) in    formula (SG-VII), preferably the molecular weight is 1500 to 3500,    more preferably 3000; and preferably Boc and OH or    N-hydroxysuccinimid, or Fmoc and N-hydroxysuccinimid.

The OH connected via the bond denoted with (****) makes SGroup, thecarboxylic acid group or the OH can also be used in its preactivatedform, as outlined further above.

As an illustration, when compound SGroup is compound SGroup1 or SGroup2with Alloc, then the compound of formula (I-SG) is the compound offormula (I-SGroup1-Fmoc) or (I-SGroup2-Alloc) respectively;

wherein

-   m1, m5, PG2, Xaa1, Xaa2 and ResinA have the same definition as    above, also with all their preferred embodiments.-   The compound HGroup is the precursor of HG, with HG being as defined    above, also with all its preferred embodiments, and has two reactive    functional groups HGroup-FunSiteN and HGroup-FunSiteC, as explained    above in the context of method(DKP-L-ResinA).

The reactive functional group HGroup-FunSiteN preferably is OH or NH₂,more preferably NH₂. The reactive functional group HGroup-FunSiteN ispresent in the protected or coupled state as a connecting group CG-HG,CG-HG being —O—, —S— or —N(H)—.

-   -   The reactive functional group HGroup-FunSiteC is usually        unprotected and is the coupling site in the respective coupling        reaction. Preferably, the reactive functional group        HGroup-FunSiteC is a carboxylic acid group. After this coupling        reaction, any protecting group of the reactive functional group        HGroup-FunSiteN is cleaved in order to make the reactive        functional group HGroup-FunSiteN available for the next coupling        reaction.

Preferably, compound HGroup is selected from the group consisting ofcompound of formula (HGroupF-I), compound of formula (HGroupF-II),compound of formula (HGroupF-III), compound of formula (HGroupF-IV),compound of formula (HGroupF-V) and compound of formula (HGroupF-VI),

wherein

-   R1, R2, R3, R4, R10 and R11 are as defined above, also with all    their preferred embodiments,-   s1-1, s2, s3, s4 and s6 are as defined above, also with all their    preferred embodiments,-   s5-1 is as defined above, also with all its preferred embodiments,-   s1-2, s5-2 and s5-3 are as defined above, also with all their    preferred embodiments,-   T1-1 is as defined above, also with all its preferred embodiments,-   T1-2 and T5-1 are as defined above, also with all their preferred    embodiments;-   H-PGHG is hydrogen in case of compound HGroup being selected from    the group consisting of compound of formula (HGroupF-I) in case that    T1-1 is O, compound of formula (HGroupF-IV) and compound of formula    (HGroupF-V);-   H-PGHG is a protecting group PGHG in case of compound HGroup being    selected from the group consisting of compound of formula    (HGroupF-I) in case that T1-1 is NH, compound of formula    (HGroupF-II), compound of formula (HGroupF-III) and compound of    formula (HGroupF-VI).

Since the compound HGroup is derived from the HG, with HG being asdefined above, also with all its preferred embodiments,

-   the (*) in any of the above definitions of HG denoting, in case of    compound HGroup, a bond between a HG and a hydrogen in case of HG    being a handle group selected from the group consisting of handle    group of formula (HGF-I) in case that T1-1 is O, handle group of    formula (HGF-IV) and handle group of formula (HGF-V);-   the (*) in any of the above definitions of HG denoting, in case of    compound HGroup, a bond between a HG and a protecting group PGHG in    case of HG being a handle group selected from the group consisting    of handle group of formula (HGF-I) in case that T1-1 is NH, handle    group of formula (HGF-II), handle group of formula (HGF-III) and    handle group of formula (HGF-VI); and-   the (**) in any of the above definitions of HG denoting in case of    compound HGroup a bond between HG and an OH.

This means, that in case, that the (*) in any of the above definitionsof HG denotes, in case of compound HGroup, a bond between a HG and aprotecting group PGHG, than the protecting PGHG is cleaved from HG afterthe coupling of compound HGroup.

Therefore, especially preferred compounds HGroup are derived from thegroup consisting of compound of formula (HG-Ia), compound of formula(HG-Ib), compound of formula (HG-Ic), compound of formula (HG-Id),compound of formula (HG-II), compound of formula (HG-III), compound offormula (HG-IVa), compound of formula (HG-IVb), compound of formula(HG-Va), compound of formula (HG-Vb) and compound of formula (HG-VI);with PGHG being as defined above, also with all its preferredembodiments.

The protection group of the side chain of Xaa2 residue is different fromPG2 and is cleavable under conditions different from those needed tocleave PG2 from Xaa2, and different from those needed to cleave Xaa1from ResinA.

Further subject of the invention is a method(G) for the preparation of acompound of formula (pDKP), with the compound of formula (pDKP) being asdefined above, also with all its preferred embodiments, characterized bya cleaving reaction (pDKP-Cleav) of a protecting group CPG from acompound of formula (pDKP-CPG);

wherein

-   H-PGHG, HG, n, SG, PG2, Xaa1 and Xaa2 have the same definition as    above, also with all their preferred embodiments;-   CPG is protecting group conventionally used in peptide chemistry for    protecting the carboxylic acid group of an amino acid or of the    C-terminus of a peptide, and is selected from the group consisting    of basic cleavable type protecting groups, acid cleavable type    protecting groups and reductively cleavable type protecting groups;-   with the proviso, that the protecting group CPG is a protecting    group different from PG2 and that CPG is cleavable under reaction    conditions different from those needed to cleave PG2 from Xaa2,-   and with the proviso, that the reaction conditions used to cleave    the protecting group CPG do not cleave PG2 from Xaa2;-   and in case, that H-PGHG is a protecting group PGHG, than-   with the proviso, that the protecting group CPG is a protecting    group different from PGHG and that CPG is cleavable under reaction    conditions different from those needed to cleave PGHG from HG,-   and with the proviso, that the reaction conditions used to cleave    the protecting group CPG do not cleave PGHG from HG.-   CPG is an embodiment of C-PG.

Compound of formula (pDKP-CPG) is an embodiment of above defined DKP-L.

Preferably, CPG is selected from the group consisting of allyl ester,Bzl (benzyl, also abbreviated with Bn) ester, Fm (9-fluorenylmethyl)ester, Me (methyl) ester, Et (ethyl) ester, Trt (triphenylmethyl ortrityl or Tr) ester, tBu ester and SiEt₃ (triethylsilyl ester or TES).

-   Preferably, in case of protecting group CPG being a basic cleavable    type protecting group, the protecting group CPG is selected from the    group consisting of Fm, Me and Et;    in case of protecting group CPG being an acid cleavable type    protecting group, the protecting group CPG is selected from the    group consisting of Trt, tBu and SiEt₃;-   and in case of protecting group CPG being a reductively cleavable    type protecting group, the protecting group CPG is selected from the    group consisting of allyl and Bzl.-   More preferably, CPG is Trt or Bzl.-   Preferably, PG2 is Fmoc and    -   CPG is allyl;    -   preferably, a possible PGHG is Boc.-   In another preferred embodiment, PG2 is Alloc and    -   CPG is Fmoc;    -   preferably, a possible PGHG is selected from the group        consisting of tBu, Trt, Mtt, Mmt and Ddz.-   In another preferred embodiment, PG2 is selected from the group    consisting of tBu, Trt, CI-Trt, Mtt, Mmt and Ddz; and    -   CPG is Allyl;    -   preferably, a possible PGHG is Fmoc.-   More preferably, PG2 is Trt and    -   CPG is Allyl;    -   preferably, a possible PGHG is Fmoc.

Further subject of the invention is a method(H) for the preparation of acompound of formula (pDKP-CPG), with the compound of formula (pDKP-CPG)being as defined above, also with all its preferred embodiments,method(H) comprises

-   a step (H1A) for the case that n is 0; or method(H) comprises-   a step (H3A) and a step (H3B) for the case that n is 1, with the    step (H3B) being done after the step (H3A);-   with n as defined above, also with all its preferred embodiments;-   wherein-   step (H3A) is a coupling reaction (H3A-Coup) of a compound of    formula (pDKP-PG2),

wherein

-   PG2, Xaa1, Xaa2 and CPG have the same definition as above, also with    all their preferred embodiments;-   the H denoted with (6) is the hydrogen of FG, FG being as defined    above, also with all it preferred embodiments as defined above;-   with a compound SGroup, the compound SGroup being as defined above,    also with all its preferred embodiments;    in case, that the reactive functional group SGroup-FunSiteN of    compound SGroup is protected by a protecting group PGSG, than in a    consecutive step (H-ConC) after step (H3A) the protecting PGSG is    cleaved from SG;-   with the proviso, that the protecting group PGSG is a protecting    group different from PG2 and that PGSG is cleavable under reaction    conditions different from those needed to cleave PG2 from Xaa2,-   and with the further proviso, that the reaction conditions used to    cleave the protecting group PGSG do not cleave PG2 from Xaa2;-   and with the further proviso, that the reaction conditions used to    cleave the protecting group PGSG in a step (H-ConC) do not cleave    Xaa1 from ResinA;-   providing a compound of formula (pDKP-SG);

-   wherein-   SG, PG2, Xaa1, Xaa2 and CPG have the same definition as above, also    with all their preferred embodiments;-   the H denoted with (7) is a hydrogen of the reactive functional    group SGroup-FunSiteN;    step (H3B) is a coupling reaction (H3B-Coup) of the compound of    formula (pDKP-SG) with a compound HGroup, with the compound HGroup    being as defined above, also with all its preferred embodiments;    step (H1A) is a coupling reaction (H1A-Coup) of the compound of    formula (pDKP-PG2) with the compound HGroup, with the compound    HGroup being as defined above, also with all its preferred    embodiments;    in case, that the reactive functional group HGroup-FunSiteN of    compound HGroup is protected by a protecting group PGHG, than in a    consecutive step (H-ConA) after step (H3B) or after step (H1A), the    protecting PGHG is cleaved from HG;-   with the proviso, that the protecting group PGHG is a protecting    group different from PG2 and that PGHG is cleavable under reaction    conditions different from those needed to cleave PG2 from Xaa2;-   and with the further proviso, that the reaction conditions used to    cleave the protecting group PGHG do not cleave PG2 from Xaa2;-   and with the further proviso, that the reaction conditions used to    cleave the protecting group PGHG in a step (H-ConA) do not cleave    Xaa1 from ResinA.    Method(H) is comprised in above defined method(DKP-L).-   Compound of formula (pDKP-PG2) and compound of formula (pDKP-SG) are    embodiments of intermediates of above defined method(DKP-L).

The protection group of the side chain of Xaa2 residue is different fromPG2 and is cleavable under conditions different from those needed tocleave PG2 from Xaa2, and different from those needed to cleave CPG fromXaa1.

Preferably, the above defined methods are done consecutively, method(B)is done after method(C), method(C) is done after method(D), method(D) isdone after method(E) and method(E) is done after method(F); a method(G)is optionally done before method(F) and a method(H) before a method(G);and the above defined respective compounds are in this caseintermediates in this sequence of methods for the preparation ofcompound of formula (III-H).

Further subject of the invention are following methods:

-   1. a method(A), wherein    -   the compound of formula (III-H) has been prepared by the        method(B);-   2. a method(A), wherein    -   the compound of formula (II-H) has been prepared by the        method(B); and wherein the compound of formula        (III-PGXaaC^((pc))) of method(B) has been prepared by method(C);-   3. a method(A), wherein    -   the compound of formula (III-H) has been prepared by the        method(B); and wherein the compound of formula        (III-PGXaaC^((pc))) of method(B) has been prepared by method(C);        and wherein    -   the compound of formula (II-PG2) of method(C) has been prepared        by method(D);-   4. a method(A), wherein    -   the compound of formula (III-H) has been prepared by the        method(B); and wherein    -   the compound of formula (III-PGXaaC^((pc))) of method(B) has        been prepared by method(C); and wherein    -   the compound of formula (II-PG2) of method(C) has been prepared        by method(D); and wherein    -   the compound of formula (II-XaaC^((pc))) of method(D) has been        prepared by method(E);-   5. a method(A), wherein    -   the compound of formula (III-H) has been prepared by the        method(B); and wherein    -   the compound of formula (III-PGXaaC^((pc))) of method(B) has        been prepared by method(C); and wherein    -   the compound of formula (II-PG2) of method(C) has been prepared        by method(D); and wherein    -   the compound of formula (II-XaaC⁽¹⁾) of method(D) has been        prepared by method(E); and wherein    -   the compound of formula (I-HG) of method(E) has been prepared by        method(F);-   6. a method(A), wherein    -   the compound of formula (III-H) has been prepared by the        method(B); and wherein    -   the compound of formula (III-PGXaaC(P) of method(B) has been        prepared by method(C); and wherein    -   the compound of formula (II-PG2) of method(C) has been prepared        by method(D); and wherein    -   the compound of formula (III-Xaa⁽¹⁾) of method(D) has been        prepared by method(E); and wherein    -   the compound of formula (I-HG) of method(E) has been prepared by        method(F); and wherein    -   the compound of formula (pDKP) of method(F) has been prepared by        method(G);-   7. a method(A), wherein    -   the compound of formula (III-H) has been prepared by the        method(B); and wherein the compound of formula        (III-PGXaaC^((pc))) of method(B) has been prepared by method(C);        and wherein    -   the compound of formula (II-PG2) of method(C) has been prepared        by method(D); and wherein    -   the compound of formula (II-XaaC⁽¹⁾) of method(D) has been        prepared by method(E); and wherein    -   the compound of formula (I-HG) of method(E) has been prepared by        method(F); and wherein    -   the compound of formula (pDKP) of method(F) has been prepared by        method(G); and wherein    -   the compound of formula (pDKP-CPG) of method(G) has been        prepared by method(H).        Method(A) is an embodiment of step (ii-pep) of        method(PEP-HSPPS).        Method(B) is preferably comprised in method(C-PEP).        Step (b) of method(C) is an embodiment of step (iii) of        method(C-PEP).        Step (a) of method(C) is comprised in a preferred embodiment of        step (iii) of method(C-PEP).        Method(D) is an embodiment of step (ii) of method(C-PEP).        Method(E) is an embodiment of step (i) of method(C-PEP).        Step (F4) of method(F) is an embodiment of method(X2).        Step (F3A) of method(F) is an embodiment of the step (X1-iii) of        method(X1).        Step (F3B) of method(F) is an embodiment of the step (X1-iv) of        method(X1).        Step (F1A) of method(F) is an embodiment of the step (X1-iv) of        method(X1).        Method(G) is an embodiment of method(DKP-L).-   If step (DKP-L-i), optional step (DKP-L-ii) and the step (DKP-L-iii)    of method(DKP-L) are done consecutively, then step (H1) of method(H)    is an embodiment of step (DKP-L-i), and steps (H3A) and (H3B) of    method(H) are embodiments of steps (DKP-L-ii) and (DKP-L-iii).

Therefore further subject of the invention are following methods:

-   (a) a method(PEP-HSPPS), wherein step (ii-pep) is method(A);-   (b) a method(C-PEP) comprising method(B);-   (c) a method(C-PEP), wherein step (iii) comprises step (b) of    method(C);-   (d) a method(C-PEP), wherein step (iii) comprises step (a) of    method(C);-   (e) a method(C-PEP), wherein step (ii) comprises method(D);-   (f) a method(C-PEP), wherein step (i) comprises method(E);-   (g) a method(DKP-L-ResinA), wherein method(X2) comprises step (F4)    of method(F);-   (h) a method(DKP-L-ResinA), wherein step (X1-iii) of method(X)    comprises step (F3A) of method(F);-   (i) a method(DKP-L-ResinA), wherein step (X1-iv) of method(X1)    comprises step (F3B) of method(F);-   (j) a method(DKP-L-ResinA), wherein step (X1-iv) of method(X1)    comprises step (F1A) of method(F);-   (k) a method(DKP-L) comprising method(G);-   (l) a method(DKP-L), wherein the step (DKP-L-i), the optional step    (DKP-L-ii) and the step (DKP-L-iii) are done consecutively, and    wherein step (DKP-L-i) comprises step (H1) of method(H), and steps    (DKP-L-ii) and (DKP-L-iii) comprise the steps (H3A) and (H3B) of    method(H).-   Further subject of the invention is compound of formula (III-H),    compound of formula (III-PGXaaC^((pc))), compound of formula    (II-PG2), compound of formula (II-H), compound of formula    (II-XaaC⁽¹⁾), compound of formula (I-HG), compound of formula    (I-SG), compound of formula (pDKP), compound of formula (pDKP-CPG)    and compound of formula (pDKP-SG);-   with the compound of formula (III-H), the compound of formula    (III-PGXaaC^((pc)), the compound of formula (II-PG2), the compound    of formula (II-H), the compound of formula (II-XaaC⁽¹⁾), the    compound of formula (I-HG), the compound of formula (I-SG), the    compound of formula (pDKP), the compound of formula (pDKP-CPG) and    the compound of formula (pDKP-SG) as defined above, also with all    their preferred embodiments.

Compound of formula (I-HG) is one embodiment of DKP-L-ResinA.

The compounds of formula (I-PG2) are known compounds and can be preparedby conventional SPPS with subsequent deprotection of the side chain ofthe Xaa2 residue.

The compounds of formula (pDKP-PG2) are known compounds and can beprepared by conventional coupling of an N-terminally and side chainprotected amino acid PG2-Xaa2 with a C-terminally protected amino acidXaa1-CPG, and with subsequent deprotection of the side chain of the Xaa2residue.

Further subject of the invention are

-   (u1) the use of a compound selected from the group consisting of    compound of formula (III-H), compound of formula    (III-PGXaaC^((pc))), compound of formula (II-PG2), compound of    formula (II-H), compound of formula (II-XaaC⁽¹⁾), compound of    formula (I-HG), compound of formula (I-SG), compound of formula    (pDKP), compound of formula (pDKP-CPG) and compound of formula    (pDKP-SG); or the use of compound of formula (pDKP) or of compound    of formula (pDKP-CPG) as a DKP-PG forming linker;    -   in peptide chemistry;    -   for the preparation of a peptide; or    -   in a method for the preparation of a peptide; or    -   in a step of a method for the preparation of a peptide; or    -   in a peptide coupling reaction; or    -   in SPPS for the preparation of a peptide; or    -   in HSPPS for the preparation of a peptide;-   (u2) the use of a compound of formula (III-PGXaaC^((pc))) for the    preparation of a compound of formula (III-H);-   (u3) the use of a compound of formula (II-PG2) for the preparation    of a compound of formula (II-H) or of a compound of formula    (III-PGXaaC^((pc));-   (u4) the use of a compound of formula (II-H) for the preparation of    a compound of formula (III-PGXaaC^((pc)));-   (u5) the use of a compound of formula (II-XaaC⁽¹⁾ for the    preparation of a compound of formula (III-PGXaaC^((pc))));-   (u6) the use of a compound of formula (I-HG) for the preparation of    a compound of formula (II-XaaC⁽¹⁾);-   (u7) the use of a compound of formula (I-SG) for the preparation of    a compound of formula (I-HG);-   (u8) the use of compound of formula (pDKP) for the preparation of a    compound of formula (I-HG);-   (u9) the use of a compound of formula (pDKP-CPG) for the preparation    of a compound of formula (pDKP);-   (u9) the use of a compound of formula (pDKP-SG) for the preparation    of a compound of formula (pDKP-CPG).-   (u10) the use of a compound of formula (pDKP-PG2) for the    preparation of a compound of formula (pDKP-SG) or for the    preparation of a compound of formula (pDKP-CPG).-   (u11) the use of a compound of formula (I-PG2) for the preparation    of a compound of formula (I-SG) or for the preparation of a compound    of formula (I-HG);-   (u12) the use of a compound selected from the group consisting of    compound of formula (III-PGXaaC^((pc))), compound of formula    (II-PG2), compound of formula (II-H), compound of formula    (II-XaaC⁽¹⁾), compound of formula (I-HG), compound of formula    (I-SG), compound of formula (pDKP), compound of formula (pDKP-CPG)    and compound of formula (pDKP-SG), for or in the preparation of    compound of formula (III-H);-   (u13) the use of the residue DKP-PG or the residue of formula    (III-res) as a protecting group, preferably as a protecting group in    peptide chemistry, preferably as a C-terminal protecting group.-   with these compounds and residues as defined above, also with all    their preferred embodiments.

The present invention allows cleavage of the C-terminal fragment of thedesired peptide from the supporting resin after its preparation withSPPS and its C-terminal protection in one single step, and this providesin comparison to the conventional two step procedure, i.e. firstlycleavage from supporting resin and secondly protecting of theC-terminus, for higher yield due to more complete reaction and/or lessundesired side-reactions, no need for additional process step, reactiontime, reagents and equipment, i.e. faster and more economic overallprocedure, less solubility issues, as protected peptides are often notvery well soluble in organic solvents, and less risk of epimerization ofthe peptide.

Also for hybrid synthesis of a peptide amide, the invention provides fora method of preparation the C-terminal fragment, which omits a multistep approach such as first preparing the C-terminal fragment startingwith the amino acid of position 2 from the C-terminus and addition ofthe amino acid in position 1 from the C-terminus in separate steps.

The method avoids partial loss of side chain protecting groups, avoidsthe risk of epimerization of the peptide due to the coupling of theamino carboxamide, it needs less process steps, time, reagents andequipment, and the method thereby has a higher yield due to morecomplete reaction, less process steps and less undesired side-reactions.

Further advantage of the invention is the use of Rink amide handle inHSHSPPS. Usually, when cleaving a peptide after SPPS from a Rink amidehandle modified resin, total deprotection of the side chains of thepeptide occurs simultaneously, thereby the Rink amide handle is usuallynot used in HSHSPPS. Due to the invention, the Rink amide handle remainsin the C-terminal DKP linker group, thereby making the peptide fragmentusable as C-terminal fragment in HSPPS, and the Rink amide handlecomprising C-terminal linker group is finally cleaved together with theside chain protecting groups in the final step providing the targetpeptide.

Another advantage of the method of the invention is the possibility toproduce side chain protected fragments C-PEP to be used in HSPPS, whichhave an amide as C-terminus. Such peptides are usually prepared usingthe Sieber Amide resin comprising the Sieber handle. Specific cleavageconditions in case of the use of the Sieber Amide resin are necessary tocleave the peptide from the resin without side chain deprotection,whereas common cleavage condition lead also in case of the Sieber Amideresin to at least partial deprotection of some of the side chains. Whenusing the method of the invention for the preparation of a fragmentC-PEP having an amide at its C-terminus, the applicability of Sieberhandle is broadened, since the Sieber handle is used as the handle groupHG in the linker and the cleavage of the peptide from the handle groupof the linker is postponed to the very last step in HSHSPPS and can bedone under global deprotection conditions, whereas the necessarycleavage from the resin providing the DKP linker group comprisingpeptide fragment used as C-terminal fragment in HSPPS is done underconditions different from those which would cleave side chain protectinggroups, and these conditions used to cleave the linker from the resin bysimultaneous generation of the DKP linker group do not lead to thecleavage of the peptide from the Sieber handle moiety nor to side chaindeprotection. This broadens the scope of use of the Sieber handle withrespect to side chain protected peptide fragment synthesis withoutnecessitating specific cleavage conditions.

In case of Xaa1 being a compound of formula (HypX), the solubility ofthe peptidyl fragment comprising the DKP linker group can be greatlyenhanced by using a solubility enhancing substituent of formula (Sub-R8)for R8, and therefore the HSPPS with C-terminal fragments with longamino acid chains becomes possible, which is necessary, if targetpeptides having many amino acid residues are to be synthesized from manyfragments, and the first C-terminal fragment comprising the DKP linkergroup is consecutively coupled to the successive PEP-N fragments byrepetitive HSPPS.

Furthermore, the possibility of using a spacer group SG can provide forhigher solubility of the fragment C-PEP in HSPPS, and therefore theHSPPS with C-terminal fragments with long amino acid chains becomespossible, which is necessary, if target peptides having many amino acidresidues are to be synthesized from many fragments, and the firstC-terminal fragment comprising the DKP linker group is consecutivelycoupled to the successive PEP-N fragments by repetitive HSPPS.

Furthermore, the spacer group SG removes spatially the reaction centre,which is the N-terminus to which the amino acids building blocks aresequentially coupled, during SPPS away from the solid support, therebythe accessibility of the reaction centre for the dissolved reagents,additives, amino acids building blocks and so on is greatly improved.

EXAMPLES Abbreviations and Raw Materials

The following abbreviations and raw materials have been and are used inthe following, if not otherwise stated.

-   Ac acetyl-   ACN acetonitrile-   CTC resin 2-chlorotrityl chloride resin (beads, 100 to 200 mesh (the    mesh is measured according to American Society for Testing and    Materials international, ASTM international), 1.57 mmol/g, “mmol/g”    means “mmol active sites/g resin”)-   DBF-Adduct 1-(9H-fluoren-9-ylmethyl)piperidine-   DCM dichloromethane-   DIEA diisopropylethylamine-   DIPE diisopropyl ether-   DIPCDI N,N′-diisopropylcarbodiimide-   DMAP 4-dimethylaminopyridin-   DMB 1,3-dimethoxybenzene-   DMF N,N-dimethylformamide-   eq equivalent(s)    -   eq refers to the mol-equivalents, with regard to the mol of        reactive sites of the resin, if not mentioned otherwise-   Fmoc-Rink-OH    4′-{(R,S)-alpha-[1-(9-Fluorenyl)methoxycarbonylamino]-2,4-dimethoxybenzyl}-phenoxyacetic    acid, also called    p-{(R,S)-a-[1-(9H-Fluoren-9-yl)-methoxyformamido]-2,4-dimethoxybenzyl}-phenoxyacetic    acid, also called Fmoc-Rink amide handle    -   Fmoc-Rink-OH is the handle group of formula (HG-Ia), wherein the        bond denoted with (*) is connected to the        9-Fluorenylmethyloxycarbonyl group of Fmoc and the bond denoted        with (**) to OH.    -   Fmoc-Rink-OH is the compound of formula (HGroup-Ia) with PGHG        being Fmoc.-   Fmoc-Ramage-OH    [R,S]-2-{[5-(9-Fluorenylmethyloxycarbonylamino)-dibenzo[a,d]cycloheptane-2-yl]oxy}-acetic    acid, also called Fmoc-Suberol, CAS 212783-75-0, also called    Fmoc-Ramage handle    -   Fmoc-Ramage-OH is the handle group of formula (HG-VI), wherein        the bond denoted with (*) is connected to the        9-Fluorenylmethyloxycarbonyl of Fmoc and the bond denoted with        (**) to OH.    -   Fmoc-Ramage-OH is the compound of formula (HGroup-V1) with PGHG        being Fmoc.-   Fmoc-TTDS-OH    [N-1[9-Fluorenylmethoxycarbonyl]-1,13-diamino-4,7,10-trioxatridecan-succinamic    acid, CAS 172089-14-4 TTDS is the spacer group of formula (SG-1)    wherein m1 is 3. Fmoc-TTDS-OH is the compound SGroup1 wherein m1 is    3.-   HMPA 4-hydroxymethylphenoxyacetic acid, CAS 68858-21-9 HMPA is the    handle group of formula (HG-IVa), wherein the bonds denoted with (*)    and (**) are connected to OH.    -   HMPA is the compound of formula (HGroup-IVa).-   HCTU 2-(6-chloro-1H-benzotriazole-1l-yl)-1,1,3,3-tetramethylaminium    hexafluorophosphate-   HMPS resin hydroxymethylpolystyrene resin (beads, of from 100 to 200    mesh (the mesh is measured according to American Society for Testing    and Materials international, ASTM international), 0.98 mmol/g,    “mmol/g” means “mmol active sites/g resin”)-   HOBt 1-hydroxybenzotriazole. water content ca. 12% (w/w) min    minute(s)-   MW molecular weight-   Oxyma ethyl 2-cyano-2-(hydroxyimino)acetate-   PVDF polyvinylidene fluoride-   RP-HPLC reverse phase high-performance liquid chromatography-   RP-HPLC-ESMS reverse phase high-performance liquid chromatography    electrospray mass spectrometry-   resin loading mmol of peptide per g of resin-   RT room temperature-   s second(s)

Sequence Abbreviations T20 Ac[T20-1-36]NH₂. This Sequence is RegisteredUnder CAS 159519-65-0. T20C H[T20-27-36]NH₂ BocT20N Boc[T20-17-26]OHHT20N H[T20-17-26]OH HT20F H [T20-17-36]NH₂

TIS triisopropylsilaneUV ultraviolet

The sequence listing links the abbreviations of the sequences to thesequences. E.g., [T20-1-36] is the sequence of the T20 peptideconsisting of the amino acid residues 1 to 36, with the left numberrepresenting the N-terminal amino acid residue and the right numberrepresenting the C-terminal amino acid residue.

Methods Description A) Determination of Resin Loading

The resin loading was quantified by Fmoc group determination measuringthe UV absorbance at 290 nm. UV absorbance measures were carried out ona Shimadzu UV-Vis recording spectrophotometer (UV-2501 PC).

Step 1 Collect the Solution from Fmoc Group Deprotection:

The Fmoc group was removed as described in the examples. In a V_(A)-m1glass volumetric flask A the resulting solutions from the Fmoc groupdeprotection were collected. The V_(A)-ml glass volumetric flask A wastotally filled with DMF.

Step 2 Diluted Solution for UV Measurements:

A V_(C)-ml aliquot from VA-ml glass volumetric flask A was transferredto another V_(B)-ml glass volumetric flask B and then it was totallyfilled with DMF. Three different diluted V_(B)-ml solutions were prepareand measured by UV spectroscopy at 290 nm to have representativeabsorbance values.

Step 3 Quantification by UV Spectrophotometry:

A UV quartz cell of 1 cm path length was filled with DMF (referencesolution), and placed into the spectrophotometer at 290 nm (maximumabsorbance wavelength of dibenzofulvene) to obtain the zero. The UV cellwas washed twice with the diluted solution B and then filled with thissolution and its absorbance was measured at 290 nm.

Finally, the resin loading is calculated following the equation:

$\frac{\left\lbrack {A\; 290 \times {cell}\mspace{14mu} {length} \times V_{A} \times V_{B}} \right\rbrack}{\left\lbrack {{epsilon}\; 290 \times g\mspace{20mu} {of}\mspace{14mu} {resin} \times V_{C}} \right\rbrack}$

A290: measured absorbance valuecell length: 1 (cm)V_(A): volume of the glass volumetric flask A (ml)V_(B): volume of the glass volumetric flask B (ml)V_(C): aliquot volume transferred from A to B (ml)epsilon290: molar extinction coefficient of dibenzofulvene at 290 nm;5800 M⁻¹ cm⁻¹

B) Characterization by RP-HPLC and RP-HPLC-ESMS Analysis B1) RP-HPLCAnalysis

Analytical RP-HPLC was carried out on a Waters instrument comprising aseparations module (Waters 2695), an automatic injector (Waters 717 autosampler), and a UV photodiode array detector (Waters 2998), and lineargradients of mobile phase B into mobile phase A were used and arespecified in each case.

Step 1 Sample preparation:Mobile Phase A: 0.045% (v/v) aqueous TFAMobile Phase B: 0.036% (v/v) TFA in ACN

A specific amount of sample in the range of from 0.5 to 1 mg wasdissolved in ca. of from 0.5 to 1 ml of a mixture of H₂O/ACN (1:1,(v/v)), the solution was filtered through a 0.45 micrometer size pore, 4mm diameter PVDF hydrophobic filter.

Step 2 Chromatography Conditions:

Column: SunFire C18, 3.5 micrometer, 4.6×100 mm

Oven: RT

Flow rate: 1.0 ml/minDetector wavelength: 220 nmGradient run time: 8 minGradient composition: x to y % (v/v) of mobile phase B as specified inthe examples

Prior to the injection, the column was conditioned at initial conditionsfor 3 min, and after each run the column was washed with ACN for 3 min.

Step 3 Chromatographic Profile Analysis:

Measure of the area of all chromatography peaks related with theproducts from the synthesis. The areas proportion is taken as apercentage of purity of the expected products.

B2) RP-HPLC-ESMS Analysis

Analytical RP-HPLC-ESMS was performed on a Waters Micromass ZQspectrometer comprising a separations module (Waters 2695), an automaticinjector (Waters 717 auto sampler), and a UV photodiode array detector(Waters 2998), and linear gradients of mobile phase B into mobile phaseA were used.

Step 1 Sample preparation:Mobile Phase A: 0.1% (v/v) aqueous formic acidMobile Phase B: 0.07% (v/v) formic acid in ACN

A specific amount of sample in the range of 0.5 to 1 mg was dissolved inca. of from 0.5 to 1 ml of a mixture of H₂O/ACN (1:1 (v/v)), thesolution was filtered through a 0.45 micrometer size pore, 4 mm diameterPVDF hydrophobic filter.

Step 2 Chromatography-Mass Spectrometry Conditions:

Column: SunFire C18, 3.5 micrometer, 2.1×100 mm

Oven: RT

Flow rate: 0.3 ml/minDetector wavelength: 220 nmGradient run time: 8 minGradient composition: x to y % (v/v) of mobile phase B as specified inthe examplesMass range m/z (positive ion mode):500 to 2500 Da

Prior to the injection, the column was conditioned at initial conditionsfor 3 min, and after each run the column was washed with ACN for 3 min.

Step 3 Chromatography-Mass Spectrometry Analysis

The UV and MS spectres for each peak were analyzed to determine themolecular ion mass for each peak.

B3) RP-HPLC Analysis

Analytical RP-HPLC was carried out on a Agilent 1100 Series instrumentcomprising a separations module, an automatic injector, and a UVphotodiode array detector, and gradients of mobile phases C and D.

Step 1 Sample Preparation:

Mobile Phase D: 0.1% v/v aqueous TFAMobile Phase C: 0.085% (v/v) TFA in ACN

A specific amount of sample in the range of from 0.5 to 1 mg wasdissolved in ca. of from 0.5 to 1 ml of a mixture of H₂O/ACN (1:2 (v/v))

Step 2 Chromatography Conditions:

Column: Waters X-Terra MS C18, 3.5 micrometer, 4.6×150 mm

Oven: 35° C.

Flow rate: 1.0 ml/minDetector wavelength: 220 nmGradient run time: 25 minGradient composition: 10 to 97% (v/v) of mobile phase C

Prior to the injection, the column was conditioned at initial conditionsfor 2 min, and after each run the column was washed for 2 min.

Step 3 Chromatographic Profile Analysis:

Measure of the area of all chromatography peaks related with theproducts from the synthesis.

The areas proportion is taken as a percentage of purity of the expectedproducts.

B4) RP-HPLC-ESMS Analysis

Analytical RP-HPLC-ESMS was performed on a Waters 2690, and gradients ofmobile phase B into mobile phase A were used.

Step 1 Sample Preparation:

Mobile Phase A: 0.1% (v/v) aqueous trifluoroacetic acidMobile Phase B: 0.085% (v/v) trifluoroacetic acid in ACNA specific amount of sample in the range of 0.5 to 1 mg was dissolved inca. of from 0.5 to 1 ml of a mixture of H₂O/ACN (1:1 (v/v)), thesolution was filtered through a 0.45 micrometer size pore, 4 mm diameterPVDF hydrophobic filter.

Step 2 Chromatography-Mass Spectrometry Conditions:

-   Column: Waters XTerra MS C18; 150x4.6 mm-   Oven: 35° C.-   Flow rate: 1 ml/min-   Detector wavelength: 220 nm-   Run time: 30 min-   Gradient composition: 10 to 97% (v/v) of mobile phase B for 20 min,    97% (v/v) of mobile phase B for 2 min, 97 to 10% (v/v) of mobile    phase B for 1 min, 10% (v/v) of mobile phase B for 7 min.

Prior to the injection, the column was conditioned at initial conditionsfor 3 min, and after each run the column was washed with ACN for 3 min.

Step 3 Chromatography-Mass Spectrometry Analysis

The UV and MS spectres for each peak were analyzed to determine themolecular ion mass for each peak.

Mass range m/z (positive ion mode):500 to 2500 Da

c) Ninhydrin Test

-   Preparation of Reagent Solutions-   Reagent Solution A: Phenol (40 g) is dissolved in EtOH (10 ml). A    solution of KCN (65 mg) in water (100 ml) is added to pyridine    (freshly distilled over ninhydrin, 100 ml). Both solutions stirred    for 45 min with a mixed-bed ion-exchange resin, filtered, and mixed.-   Reagent Solution B: A solution of ninhydrin (2.5 g) in absolute EtOH    (50 ml) was prepared and maintained in a light-proof container,    preferably under inert atmosphere.

Experimental Procedure

Resin is washed with DCM and 1 to 5 mg is transferred to a small glasstube. To this tube are added six drops of reagent solution A and twodrops of B. The tube was then heated at 100° C. for 3 min.

Negative test (absence of free primary amines): yellow solution andnaturally coloured resin beads.Positive test (presence of free primary amines): dark blue or purplesolution and resin beads.

D) Chloranil Test Preparation of Reagent Solution

Reagent Solution: A solution of chloranil (4.1 g) in toluene (100 ml)was prepared.

Experimental Procedure

To acetone (1 ml) small glass tube, 1 drop of reagent solution and 1drop of testing solution where added. The tube was then mixed for about10 s.Negative test (absence of piperidine): colourless to light yellowPositive test (presence of piperidine): blue or purple.

Example 1 SPPS of T20C Using an Attachment Via a Diketopiperazine GroupForming Dipeptidyl Linker to the Resin (0.1 Mmol Scale)

The SPPS was performed manually.

Method Fmoc-Gr-Rem (Fmoc-Group-Removal)

In the following examples, the Fmoc group was removed by theseprocedures:

Treatment of the resin with 20% (v/v) piperidine in DMF (1×1 min, 2×10min; 3 ml each for examples 1 to 3 and for example 7, 150 ml each forexamples 4 to 6, followed by: DMF washes (5×1 min; 3 ml each) forexamples 1 to 3 and for example 7, DMF continuous washes (600 ml) andchloranil test method D for examples 4 to 6.

Example 1.1 Attachment of the Diketopiperazine Group Forming DipeptidylLinker to the Resin a) Pre-Treatment of the HMPS Resin

HMPS resin (106.2 mg) was swelled with DCM (5×1 min; 3 ml each) and DMF(5×1 min; 3 ml each) at RT and then filtered.

b) Introduction of the First Amino Acid (D-Pro) of the DiketopiperazineGroup Forming Dipeptidyl Linker on the Resin

Fmoc-D-Pro-OH (135 mg, 4 eq) and DIPCDI (31 microliter, 2 eq) in DCM/DMF(15:1 (v/v), 2.5 ml) was added to a resin prepared according to example1.1a). Then, DMAP (4.9 mg, 0.4 eq) in DCM (0.5 ml) was added and left tostand at RT for 2 h. The incorporation of the Fmoc-D-Pro-OH was carriedout a second time following this procedure. After the preceding 2 hcoupling time, the resin was washed with DCM (5×1 min; 3 ml each) andwith DMF (5×1 min; 3 ml each). Then, the resin was capped using aceticanhydride (47 microliter, 5 eq) and DIEA (85 microliter, 5 eq) in DMF(2.5 ml) for 30 min at RT. After capping, the resin was washed with DMF(5×1 min; 3 ml each) and with DCM (5×1 min; 3 ml each). Then the Fmocgroup was removed by the method Fmoc-gr-rem.

A 0.95 mmol/g resin loading was determined by UV quantification (methoddescription A;V_(A): 100 ml, V_(B): 10 ml and V_(C): 1.6 ml). Therefore 106.2 mg ofHMPS resin represents 0.1 mmol of active sites.

C) Introduction of the Second Amino Acid (L-Lys) of the DiketopiperazineGroup Forming Dipeptidyl Linker

A mixture of Trt-L-Lys(Fmoc)-OH (198 mg, 3 eq), HOBt (50 mg, 3 eq) andDIPCDI (50 microliter, 3 eq) in DMF (2 ml) was shaken for 5 min at RT,then added to a resin prepared according to example 1.1 b). The mixturewas left to stand at RT for 16 h. No re-coupling was required accordingto the ninhydrin test (method C). Then the resin was washed with DMF(5×1 min; 3 ml each) and with DCM (5×1 min; 3 ml each). Then the Fmocgroup was removed by the method Fmoc-gr-rem.

d) Incorporation of a Rink Amide Handle

Fmoc-Rink-OH (175 mg, 3 eq), HOBt (50 mg, 3 eq) and DIPCDI (50microliter, 3 eq) in DMF (2 ml) were shaken for 5 min at RT, then addedto a resin prepared according to example 1.1c). The mixture was left tostand at RT for 1 h. No re-coupling was required according to theninhydrin test (method C). After the preceding 1 h coupling time, theresin was washed with DMF (5×1 min; 3 ml each) and with DCM (5×1 min; 3ml each). Then the Fmoc group was removed by the method Fmoc-gr-rem.

Example 1.2

T20C by SPPS

Each amino acid was reacted in a reaction cycle. The reaction steps inone reaction cycle for the incorporation of one amino acid follow thereaction cycle description (i) or (ii).

a) Reaction Cycle Description (i) The mixture of Fmoc-Xaa-OH (3 eq),HCTU (140 mg, 3 eq) and DIEA (110 microliter, 6 eq) in DMF (2 ml) wasshaken for 30 s at RT, then added to the resin prepared according to thepreceding step in the elongation sequence. The mixture was left to standat RT for 1 h. No re-coupling was required according to the ninhydrintest (method C). Then the resin was washed with DMF (5×1 min; 3 ml each)and with DCM (5×1 min; 3 ml each). Then the Fmoc group was removed bythe method Fmoc-gr-rem.

b) Reaction Cycle Description (ii)

The mixture of Fmoc-Xaa-OH (3 eq), HCTU (140 mg, 3 eq), DIEA (110microliter, 6 eq) in DMF (2 ml) was shaken for 30 s at RT, then added tothe resin prepared according to the preceding step in the elongationsequence. The mixture was left to stand at RT for 1 h. A re-coupling wascarried out using a mixture of Fmoc-Xaa-OH (3 eq), HCTU (140 mg, 3 eq),DIEA (110 microliter, 6 eq) in DMF (2 ml). This mixture was shaken for30 s at RT, and then added to the resin and left to stand at RT for 1 h.Then the resin was washed with DMF (5×1 min; 3 ml each) and the resinwas capped using acetic anhydride (47 microliter, 5 eq) and DIEA (85microliter, 5 eq) in DMF (2.5 ml) for 15 min at RT. Then the resin waswashed with DMF (5×1 min; 3 ml each) and with DCM (5×1 min; 3 ml each).Then the Fmoc group was removed by the method Fmoc-gr-rem.

C) Elongation Sequence

The first amino acid, Fmoc-³⁶Phe-OH, was coupled to a resin comprising adiketopiperazine group forming dipeptidyl linker and a Rink amide handlegroup, prepared according to example 1.1d), the following amino acidswere then coupled to the amino acid/peptide Rink amide handle groupcomprising resin prepared in the preceding step in the elongationsequence. The sequence of incorporation of the amino acids was:

Reaction cycle Fmoc-Xaa-OH description 1. Fmoc-³⁶Phe-OH (128 mg) (i) 2.Fmoc-³⁵Trp(Boc)—OH (175 mg) (i) 3. Fmoc-³⁴Asn(Trt)-OH (198 mg) (i) 4.Fmoc-³³Trp(Boc)—OH (175 mg) (i) 5. Fmoc-³²Leu-OH (117 mg) (i) 6.Fmoc-³¹Ser(tBu)—OH (127 mg) (i) 7. Fmoc-³⁰Ala-OH•H2O (110 mg) (i) 8.Fmoc-²⁹Trp(Boc)—OH (175 mg) (i) 9. Fmoc-²⁸Lys(Boc)—OH (2 times 156 mg)(ii) 10. Fmoc-²⁷Asp(tBu)—OH (137 mg) (i)

Example 1.3 Analysis—Cleavage of T20C from Rink Amide Handle Group

A small portion of peptidyl-resin (5 mg), prepared according to example1.2, was cleaved from the Rink amide handle group by treating the resinwith 1 ml of a mixture consisting of 95% (v/v) TFA, 2.5% (v/v) TIS and2.5% (v/v) H₂O for 1 h at RT. The peptide was obtained in 83% purity(RP-HPLC, method description B1, 25 to 50 of mobile phase B).

Example 1.4a Trt Protecting Group Removal of the L-Lys of theDiketopiperazine Group Forming Dipeptidyl Linker, Formation of theDiketopiperazine Residue Comprising C-Terminal Protecting Group andCleavage from the Resin

In a first step, the Trt protecting group of the L-Lys of thediketopiperazine group forming dipeptidyl linker was removed by treatinga peptidyl-resin (15 mg), prepared according to example 1.2, with 0.2%(v/v) TFA in DCM (2 ml) at RT for 2×5 min. Then, in a second step, thethus obtained from Trt deprotected peptidyl-resin was neutralized bywashing with 5% (v/v) DIEA in DCM (2 ml) at RT for 2×5 min.

The RP-HPLC analysis after the first step did not show any peptidecleaved from the resin or from the Rink amide handle group, and afterthe second step, no peptidyl-resin comprising a DKP linker group wasobserved (method description B1, 5 to 100 of mobile phase B).

After the second step, the peptide comprising the diketopiperazineresidue comprising C-terminal protecting group was obtained by treatingthe peptidyl resin from the second step with 5% (v/v) piperidine in THFat RT (5×5 min, 2 ml each). THF was removed by evaporation under vacuumand the resulting peptide comprising the diketopiperazine residuecomprising C-terminal protecting group was analyzed by RP-HPLC-ESMS(method description B2, 80 to 100 of mobile phase B, [(M+2H)/2]2+:1308.0, respectively, where M is the MW of T20C comprising the DKPlinker group. The expected product was obtained as a racemic mixture dueto the Rink amide handle group, and the observed molecular masscorresponded to the theoretically expected mass.

In order to quantify how much of the diketopiperazine group formingdipeptidyl linker had remained on the resin, the resin was treated with2 ml of a mixture consisting of 95% (v/v) TFA, 2.5% (v/v) TIS and 2.5%(v/v) H2O for 1 h at RT. The RP-HLPC analysis showed that there was lessthan 1% left of diketopiperazine group forming dipeptidyl linker in theresin (method description B1, 5 to 100 of mobile phase B).

Example 1.4b

Example 1.4a was repeated with the sole difference, that after thesecond step, the peptide comprising the diketopiperazine residuecomprising C-terminal protecting group was obtained not by treating theresin with 5% (v/v) piperidine in THF at RT (5×5 min, 2 ml each) as inexample 1.4a, but by treating with 5% (v/v) piperidine in DMF at RT (5×5min, 2 ml each). DMF was removed by co-evaporation with toluene (5×3 ml)under vacuum.

Example 1.4c

Example 1.4a was repeated with the sole difference, that after thesecond step, the peptide comprising the diketopiperazine residuecomprising C-terminal protecting group was obtained not by treating theresin with 5% (v/v) piperidine in THF at RT (5×5 min, 2 ml each) as inexample 1.4a, but by treating with 5% (v/v) pyrrolidine in THF at RT(5×5 min, 2 ml each).

Example 1.4d

Example 1.4a was repeated with the sole difference, that after thesecond step, the peptide comprising the diketopiperazine residuecomprising C-terminal protecting group was obtained not by treating theresin with 5% (v/v) piperidine in THF at RT (5×5 min, 2 ml each) as inexample 1.4a, but by treating with 5% (v/v) pyrrolidine in DMF at RT(5×5 min, 2 ml each).

DMF was removed by co-evaporation with toluene (5×3 ml) under vacuum.

In all three examples 1.4b, 1.4c and 1.4d, the same analytical resultswith RP-HPLC-ESMS analysis and with the determination of residuallyremaining diketopiperazine group forming dipeptidyl linker on the resinwere obtained as in example 1.4a.

Example 2 SPPS of T20C Using as Attachment to the Resin aDiketopiperazine Residue Comprising C-Terminal Protecting Group (5 MmolScale)

The SPPS was Performed Manually.

Example 2.1 Attachment of the Diketopiperazine Group Forming DipeptidylLinker to the HMPS Resin a) Pre-Treatment of HMPS Resin

HMPS resin (5.0977 g) was swelled with DCM (5×1 min; 50 ml each) and DMF(5×1 min; 50 ml each) at RT and then filtered.

B) Introduction of the First Amino Acid (D-Pro) of the DiketopiperazineGroup Forming Dipeptidyl Linker on the Resin.

Fmoc-D-Pro-OH (6.6 g, 4 eq) and DIPCDI (1.5 ml, 2 eq) in DCM/DMF (15:1(v/v), 100 ml) was added to a resin prepared according to example 2.1a). Then, DMAP (245 mg, 0.4 eq) in DCM (5 ml) was added and left tostand at RT for 16 h. The first amino acid was re-coupled usingFmoc-D-Pro-OH (6.6 g, 4 eq) and DIPCDI (1.5 ml, 2 eq) in DCM/DMF (15:1(v/v), 100 ml) for 5 h at RT. After coupling, the resin was washed withDCM (5×1 min; 50 ml each) and with DMF (5×1 min; 50 ml each). Then, theresin was capped using acetic anhydride (2.4 ml, 5 eq) and DIEA (4.4 ml,5 eq) in DMF (50 ml) for 1 h at RT. After capping, the resin was washedwith DCM (5×1 min; 50 ml each) and with DMF (5×1 min; 50 ml each). Thenthe Fmoc group was removed by treatment with piperidine/DMF (20% (v/v),1×1 min, 2×10 min; 50 ml each).

A 0.98 mmol/g resin loading was determined by UV quantification (methoddescription A;VA: 250 ml, VB: 50 ml, and VC: 0.44 ml). Therefore 5.0977 g of HMPSresin represents 5.0 mmol of active sites.

C) Introduction of the Second Amino Acid (L-Lys) of the DiketopiperazineGroup Forming Dipeptidyl Linker

A mixture of Trt-Lys(Fmoc)-OH (9.2 g, 3 eq), HOBt (2.3 g, 3 eq) andDIPCDI (2.3 ml, 3 eq) in DMF (100 ml) was shaken for 5 min at RT, thenadded to a resin prepared according to example 2.1b), and then themixture was left to stand at RT for 16 h. No re-coupling was required,according to the ninhydrin test (method C). Then the resin was washedwith DMF (5 ×1 min; 50 ml each) and with DCM (5×1 min; 50 ml each). Thenthe Fmoc group was removed by treatment with piperidine/DMF (20% (v/v),1×1 min, 2×10 min; 50 ml each).

d) Introduction of a Rink Amide Handle Group

A mixture of Fmoc-Rink-OH (7.9 g, 3 eq), HOBt (2.3 g, 3 eq) and DIPCDI(2.3 ml, 3 eq) in DMF (100 ml) was shaken for 5 min at RT, then added toa resin prepared according to example 2.1c), and then left to stand atRT for 16 h. No re-coupling was required, according to the ninhydrintest (method C). Then the resin was washed with DMF (5×1 min; 50 mleach) and with DCM (5×1 min; 50 ml each). Then the Fmoc group wasremoved by treatment with piperidine/DMF (20/(v/v), 1×1 min, 2×10 min;100 ml each).

Example 2.2 T20C by SPPS

Each amino acid was reacted in a reaction cycle. The reaction steps inone reaction cycle for the incorporation of one amino acid follow theReaction cycle description (iii) or (iv).

a) Reaction Cycle Description (III)

A mixture of Fmoc-Xaa-OH (3 eq), HCTU (6.2 g, 3 eq), DIEA (5.2 ml, 6 eq)in DMF (100 ml) was shaken for 30 s at RT, then added to the resinprepared according to the preceding step in the elongation sequence. Themixture was then left to stand at RT for 2 h. No re-coupling wasrequired, according to the ninhydrin test (method C). Then the resin waswashed with DMF (5×1 min; 100 ml each) and with DCM (5×1 min; 100 mleach). Then the Fmoc group was removed by treatment with piperidine/DMF(20% (v/v), 1×1 min, 2×10 min; 100 ml each).

b) Reaction Cycle Description (iv)

A mixture of Fmoc-Xaa-OH (3 eq), HOBt (2.3 g, 3 eq), DIPCDI (2.3 ml, 3eq) in DMF (100 ml) was shaken for 5 min at RT, then added to the resinprepared according to the preceding step in the elongation sequence, andthen left to stand at RT for 16 h. A re-coupling was carried out using amixture of Fmoc-Xaa-OH (3 eq), HCTU (6.2 g, 3 eq), DIEA (5.2 ml, 6 eq)in DMF (100 ml). This mixture was shaken for 30 s at RT, and then addedto the resin and left to stand at RT for 2 h. Then, the resin was washedwith DMF (5×1 min; 100 ml each) and the resin was capped using aceticanhydride (2.4 ml, 5 eq), DIEA (4.4 ml, 5 eq) in DMF (100 ml) for 1 h atRT. Then, the resin was washed with DMF (5×1 min; 100 ml each) and withDCM (5×1 min; 100 ml each). Then the Fmoc group was removed by treatmentwith piperidine/DMF (20% (v/v), 1×1 min, 2×10 min; 100 ml each).

c) Elongation Sequence

The first amino acid, Fmoc-³⁶Phe-OH, was coupled to a Rink amide handlegroup and diketopiperazine group forming dipeptidyl linker comprisingresin, prepared according to example 2.1d), the following amino acidswere then coupled to with the resulting amino acid/peptide Rink amidehandle group and diketopiperazine group forming dipeptidyl linkercomprising resin prepared in the respective preceding step in theelongation sequence. The sequence of incorporation of the amino acids isgiven in table c1):

TABLE c1 Reaction cycle Fmoc-Xaa-OH description 1. Fmoc-³⁶Phe-OH (5.8 g)(iii) 2. Fmoc-³⁵Trp(Boc)—OH (7.9 g) (iii) 3. Fmoc-³⁴Asn(Trt)-OH (8.8 g)(iii) 4. Fmoc-³³Trp(Boc)—OH (7.9 g) (iii) 5. Fmoc-³²Leu-OH (5.3 g) (iii)6. Fmoc-³¹Ser(tBu)—OH (5.6 g) (iii) 7. Fmoc-³⁰Ala-OH•H2O (4.7 g) (iii)8. Fmoc-²⁹Trp(Boc)—OH (7.9 g) (iii) 9. Fmoc-²⁸Lys(Boc)—OH (2 times 6.8g) (iv) 10. Fmoc-²⁷Asp(tBu)—OH (6.0 g) (iii)

Example 2.3 Analysis—Cleavage of T20C from Rink Amide Handle Group

A small portion of peptidyl-resin (5 mg), prepared according to example2.2, was cleaved from the Rink amide handle group by treating the resinwith 1 ml of a mixture of 95% (v/v) TFA, 2.5% (v/v) TIS, and 2.5% (v/v)H2O for 1 h at RT. The peptide was obtained in 72% purity, as determinedby analytical RP-HPLC (method description B1, 25 to 50 of mobile phaseB).

Example 2.4 Trt Protecting Group Removal of the L-Lys of theDiketopiperazine Group Forming Dipeptidyl Linker, Formation of theDiketopiperazine Residue Comprising C-Terminal Protecting Group andCleavage from the Resin

Diketopiperazine residue comprising C-terminal protecting groupformation and cleavage of a peptide fragment, prepared according toexample 2.2, from the resin (1.57 g) are brought about in analogousmanner as described in example 1.4a with the amounts of reagents andsolvent adapted to the higher amount of peptidyl-resin:

a) Trt Group Deprotection

0.5% (v/v) TFA in DCM (2×5 min; 20 ml each).

b) Neutralization

5% (v/v) DIEA in DMF (2×5 min; 20 ml each).

c) DKP Linker Group Formation

5% (v/v) piperidine in THF (5×5 min; 20 ml each).

The THF was removed under vacuum and the resulting crude was washed withpre-cooled (4° C.) Et₂O (50 ml×3). 642.5 mg of T20C comprising thediketopiperazine residue comprising C-terminal protecting group wereobtained.

Example 3 Preparation of HT20F: a) SPPS of BocT20N, b) HSPPS Couplingwith T20C Comprising the Diketopiperazine Residue Comprising C-TerminalProtecting Group, and c) Total Deprotection

A T20C comprising the diketopiperazine residue comprising C-terminalprotecting group was obtained according to example 2.4.

Example 3.1 BocT20N by SPPS

The SPPS of a BocT20N was performed manually by linear Fmoc SPPS. Onlylast Glu amino acid was Boc protected Boc-Glu(tBu)-OH.

a) Pre-Treatment of CTC Resin

CTC resin (5.0054 g) was swelled with DCM (5×1 min; 50 ml each) and DMF(5×1 min; 50 ml each) at RT and then filtered.

B) Introduction of the First Amino Acid (L-Leu) on the CTC Resin:

Fmoc-²⁶Leu-OH (1.8 g, 1 eq), DIEA (8.7 ml, 10 eq) in DCM (50 ml) wasadded to a resin, prepared according to example 3.1a), and the mixturewas left to stand at RT for 1 h. Then the resin was capped by treatmentof the resin with MeOH (0.8 microliter/mg resin; 4 ml) for 15 min at RT.After capping the resin was washed with DCM (5×1 min; 50 ml each) andwith DMF (5×1 min; 50 ml each). Then the Fmoc group was removed bytreating the resin with piperidine/DMF (20% (v/v), 1×1 min, 2×10 min; 50ml each).

After the Fmoc-²⁶Leu-OH incorporation, a 0.89 mmol/g resin loading wasdetermined by UV quantification (method description A; V_(A): 250 ml,V_(B): 50 ml, and V_(C): 0.34 ml).

c) BocT20N by SPPS

Each amino acid was reacted in a reaction cycle. The reaction steps inone reaction cycle for the incorporation of one amino acid follow thereaction cycle description (v).

C1) Reaction Cycle Description (V)

A mixture of Fmoc-Xaa-OH (3 eq), Oxyma (1.9 g, 3 eq), DIPCDI (2.3 ml, 3eq) in DMF (V1 ml as given in the table c2) was shaken for 5 min at RT,then added to the resin prepared according to the preceding step in theelongation sequence. Then the mixture was left to stand at RT for 16 h.No re-coupling was required, according to the ninhydrin test (method C).

Then, the resin was washed with DMF (5×1 min; V2 ml as given in thetable c2) and with DCM (5×1 min; V2 ml as given in the table c2).

Then the Fmoc group was removed by treating the resin withpiperidine/DMF (20% (v/v), 1×1 min, 2×10 min; V3 ml as given in tablec2).

C2) Elongation Sequence

The second amino acid, Fmoc-²⁵Glu(tBu)-OH, was coupled according to thereaction cycle description (v) to a resin prepared according to example3.1b), the following amino acids were then coupled according to thereaction cycle description (v) to the resulting amino acid/peptidylCTC-resin prepared in the preceding step of the elongation cycle. Thesequence of incorporation of the amino acids is given in table c2).

TABLE c2 Fmoc-Xaa-OH ml V1 ml V2 ml V3 1. Fmoc-²⁵Glu(tBu)—OH (5.7 g) 5050 50 2. Fmoc-²⁴Leu-OH (4.7 g) 50 50 50 3. Fmoc-²³Leu-OH (4.7 g) 50 5050 4. Fmoc-²²Glu(tBu)—OH (5.7 g) 70 70 70 5. Fmoc-²¹Gln(Trt)-OH (8.1 g)70 70 70 6. Fmoc-²⁰Glu(tBu)—OH (5.7 g) 100 70 70 7. Fmoc-¹⁹Asn(Trt)-OH(8.0 g) 100 70 70 8. Fmoc-¹⁸Lys(Boc)—OH (6.3 g) 100 70 70 9.Boc-¹⁷Glu(tBu)—OH (6.3 g) 100 70 70

Example 3.2 Analysis Cleavage of HT20N from CTC Resin

Peptidyl-resin (5 mg), prepared according to example 3.1c2), was treatedwith 1 ml of a mixture consisting of 95% (v/v) TFA, 2.5% (v/v) TIS and2.5% (v/v) H2O for 1 h at RT for cleaving the peptide from the CTC resinand for fully deprotecting the amino acid residues. The HT20N wasobtained in 85.7% purity, as determined by RP-HPLC (method descriptionB1, 5 to 100 of mobile phase B). The peptide was analysed byRP-HPLC-ESMS (method description B2, 5 to 100 of mobile phase B, [M+H]+:1244.7, where M corresponds to the fully deprotected HT20N).

Example 3.3 BocT20N Side Chain Protected

Peptidyl resin (1.094 g), prepared according to example 3.1 c2), wastreated with 1% (v/v) TFA in DCM (5×1 min; 50 ml each) at RT, all 5mixtures were poured into H2O (20 ml). Then, this aqueous mixture wasevaporated and the crude was lyophilized. The fully side chain protectedBocT20ON was obtained (510 mg) and was analysed by RP-HPLC-ESMS (methoddescription B2, 95 to 100 of mobile phase B, [M+H]+: 2153.8). No partialdeprotection of the fully side chain protected BocT20N was observed byRP-HPLC-ESMS (method description B1, 50 to 100 of mobile phase B).RP-HPLC showed one peak, purity was 85.7% (method description B2, 95 to100 of mobile phase B).

Example 3.4 HT20F by HSPPS a) HSPPS Coupling Between the T20C Comprisingthe Diketopiperazine Residue Comprising C-Terminal Protecting Group andBocT20N

Side chain protected BocT20N (10 mg, 4.6 micromol), prepared accordingto example 3.3, and HOBt (2.2 mg, 3 eq) were dissolved in DCM (350microliter) and DIPCDI (2.2 microliter, 3 eq) was added to the mixture.The mixture was shaken for 5 min at RT. The mixture was added to asolution of T20C comprising the diketopiperazine residue comprisingC-terminal protecting group (12 mg, 4.6 micromol), prepared according toexample 2.4, in DCM (350 microliter). The resulting mixture was stirredat RT for 16 h. The coupling was monitoring by RP-HPLC analysis (methoddescription B 1, 95 to 100 of mobile phase B), total conversion wasobserved after 16 h.

Solvent was evaporated under vacuum resulting in the fragmentBoc[T20-17-36]connected C-terminally to the diketopiperazine residuecomprising C-terminal protecting group.

b) HT20F by Total Deprotection

A fragment Boc[T20-17-36]connected C-terminally to the diketopiperazineresidue comprising C-terminal protecting group (1 mg), preparedaccording to example 3.4a), was treated with 1 ml of a mixture of 92.5%(v/v) TFA, 2.5% (v/v) TIS and 5% (v/v) DMB for 1 h at RT. In order toremove the resulting N-carboxy groups on the side chains of the Trpresidues, 0.5% (v/v) aqueous NH3 (1 ml) were added and the mixture wasleft to stand for 16 h at RT to obtain the fully deprotected HT20F with60.2% purity, as determined by RP-HPLC (method description B1, 30 to 40of mobile phase B). RP-HPLC-ESMS showed the target peptide (methoddescription B2, 30 to 40 of mobile phase B) with [(M+2H)/2]2+: 1290.0,where M is the MW of HT20F.

Example 4 Preparation of Compound of Formula (Ex-4) by SPPS

Ac-Tyr(OtBu)-His(Trt)-Ala-OH  (ex-4)

a) Pre-Treatment of CTC Resin

CTC resin (20.4 g) was swelled with DCM (1 h; 200 ml) at RT and thenfiltered.

b) Introduction of the First Amino Acid (Fmoc-Ala-OH) on the CTC Resin

Fmoc-Ala-OH (12.65 g, 1.2 eq), DIEA (14.90 g, 3.6 eq) in DCM (160 ml)was added to a resin prepared according to example 4a), the mixture wasleft to stand at RT for 2 h, and then filtered. The resin was treatedwith DIEA/MeOH (10% (v/v), 200 ml) and DMF (40 ml) for 1 h at RT andthen filtered. Then the Fmoc group was removed according to methodFmoc-gr-rem. After the Fmoc-Ala-OH incorporation, a 0.97 mmol/g resinloading was calculated.

c1) Incorporation of Amino Acids by SPPS

Starting with a resin prepared according to example 4.b), each aminoacid, respectively 1. Fmoc-His(Trt)-OH and 2. Fmoc-Tyr(tBu)-OH, wereincorporated following the reaction cycle description example 4 c2).

c2) Cycle Description

A mixture of the respective Fmoc-Xaa-OH (1.5 eq), HOBt (9.2 g, 2.25 eq),DIPCDI (9.31 ml, 2.25 eq) in DMF (103 g) was stirred for 5 min at RT,then added to a resin prepared according to example 4 b), and then leftto stand at RT for 45 min. Then DIPCDI (4.66 ml, 1.25 eq) was added andthe mixture left to stand at RT for 45 min. No re-coupling was required,according to the ninhydrin test (method C). The resin was washed withDMF (3×5 min; 110 ml each). Then the Fmoc group was removed according tomethod Fmoc-gr-rem. All piperidine was removed according to thechloranil test (method D).

d) Cleavage from the Resin

The resin prepared according to example 4 c1) was washed with 2% (w/w)TFA in DCM (4×15 min; 150 g each) at approx. 10° C. Then the resin waswashed with EtOH/DCM (20% (w/w), 3×3 min; 120 g each) at RT 10° C. Thecombine solution was concentrated by co-evaporation with EtOH underreduced pressure (1×40 g).

e) Isolation

To the solution (107.60 g) obtained according to example 4d), water (800g) was added. The resulting mixture was filtered and the solid washedwith water (3×3 min; 80 g each) and DIPE (3×2 min; 120 ml each).

The solid was dried at 30° C. under reduced pressure to obtain 20.80 gof compound of formula (ex-4) as white powder with a purity of 94.7% asdetermined by RP-HPLC (method B3).

Example 5 Attachment of the Diketopiperazine Group Forming DipeptidylLinker to the HMPS Resin, Use of a Ramage Handle Group, and Preparationof Compound of Formula (Ex-5i) a) Pre-Treatment of HMPS Resin

HMPS resin (5.0 g) was swelled with DCM (5×1 min; 150 ml each) and DMF(5×1 min; 150 ml each).

b) Introduction of the First Amino Acid on the Resin.

The respective amino acid (N(Me)Phe-OH or Fmoc-D-Pro-OH) and DIPCDI(2.43 g, 3.5 eq) in DCM/DMF (15:1 (v/v), 125 ml) was added to a resinprepared according to example 5a). Then, DMAP (0.27 g, 0.4 eq) in DCM(25 ml) was added and left to stand at RT for 3 or 4 h. The resin waswashed with DCM (5×1 min; 150 ml each) and with DMF (5×1 min; 150 mleach). Then, the resin was capped using acetic anhydride (2.81 g, 5 eq)and DIEA (3.56 g, 5 eq) in DMF (125 ml) for 30 min at RT. After capping,the resin was washed with DMF (5×1 min; 150 ml each). Then the Fmocgroup was removed according to method Fmoc-gr-rem. A 1.10 mmol/g resinloading was calculated, therefore 5.0 g of HMPS resin represents 5.5mmol of active sites.

b1) Procedure according to example 5 b) with Xaa being N(Me)Phe-OH (4.86g, 2.2 eq) and left to stand at RT for 4 h.b2) Procedure according to example 5 b) with Xaa being Fmoc-D-Pro-OH(7.45 g, 4.0 eq) and left to stand at RT for 3 h.

C) Introduction of the Second Amino Acid

A mixture of Trt-Lys(Fmoc)-OH (11.26 g, 2 eq), HOBt (4.24 g, 3 eq) andDIPCDI (3.49 g, 3 eq) in DMF (100 ml) was stirred for 5 min at RT, thenadded to a resin prepared according to example 5 b1). The mixture wasstirred at RT for 17 h.

Then, the resin was washed with DMF (5×1 min; 150 ml each), and the Fmocgroup was removed according to method Fmoc-gr-rem.

d) Incorporation of Amino Acids by SPPS

Starting with a resin prepared according to example 5 c), the handlegroup and the respective amino acids, i.e. 1. Fmoc-Ramage-OH, 2.Fmoc-Leu-OH, 3. Fmoc-Ala-OH and 4. Fmoc-Phe-OH, were incorporatedfollowing the reaction cycle description (vi).

e) Reaction Cycle Description (vi)

A mixture of the handle group or of the respective amino acidsFmoc-Xaa-OH (2 eq), HOBt (4.24 g, 3 eq), DIPCDI (3.49 g, 3 eq) in DMF(100 ml) was stirred for 5 min at RT, then added to the resin preparedaccording to according to example 5 c) (in case of the handle group) andthen d) (in case of the amino acids) respectively, and then left tostand at RT for 1 h. The resin was washed with DMF (5×1 min; 150 mleach), and additionally with DCM (5×1 min; 150 ml each) when Xaa wasFmoc-Phe-OH. The resin was washed further with DMF (1×1 min; 150 ml) atRT when Xaa was Fmoc-Ala-OH. The Fmoc group was removed according tomethod Fmoc-gr-rem except for the last Xaa, the Fmoc-Phe-OH.

f) Analysis—Cleavage of the Peptide from the Handle Group and TherebyAlso from the Resin

A small portion of resin (5 mg) obtained according to example 5 d) wastreated with 1 ml of a mixture of 95% (v/v) TFA, 2.5% (v/v) TIS and 2.5%(v/v) water for 1 h at RT. A compound of formula (ex-5f) was obtained in86.4% purity, as determined by analytical RP-HPLC (HPLC-method B3).

Fmoc-Phe-Ala-Leu-NH₂  (ex-5f)

g) Trt Protecting Group Removal of the L-Lys of the Linker, Formation ofthe Diketopiperazine Residue Comprising C-Terminal Protecting Group andCleavage from the Resin

Formation of the diketopiperazine residue comprising C-terminalprotecting group and cleavage of the DKP-peptide from the resin preparedaccording to example 5d), are brought about in analogous manner asdescribed in example 1.4a with the amounts of reagents and solventadapted to the amount of peptidyl-resin:

g1) Trt Group Deprotection

Treatment of the compound prepared according to example 5 d) with 0.2%(v/v) TFA in DCM (2×5 min, 100 ml each).

g2) Neutralization

Then treatment with 5% (v/v) DIEA in DCM (2×5 min; 100 ml each) and DCM(2×1 min; 50 ml each). HPLC was used to ensure that no product remainedin the liquid phases (HPLC-method B3).

g3) Formation of the Diketopiperazine Residue Comprising C-TerminalProtecting Group and Cleavage from the Resin

Then treatment with 5% (v/v) piperidine in THF (5×5 min, 100 ml each).Then, the resin was washed with THF (3×1 min; 100 ml each). HPLC wasused to ensure that the product was in the combined liquid phase(HPLC-method B3).

The THF was removed by co-evaporation with toluene (3×150 ml) undervacuum. 1.96 g of a white solid of a mixture of DBF-Adduct and compoundof formula (ex-5g4) were obtained. Method B4 gave the expected mass.

g4) DBF-Adduct Removal

The white solid prepared according to example 5 g3) was washed with DIPE(1×2 min, 50 ml; 3×2 min, 10 ml each). 223.7 mg of compound of formula(ex-5g4) were obtained as a white solid.

h) Analysis—Cleavage of the Peptide from the Diketopiperazine ResidueComprising C-Terminal Protecting Group

A small portion of compound of formula (ex-5g4) (5 mg), preparedaccording to example 5 g4), was deprotected by treatment with 1 ml of amixture of 95% (v/v) TFA, 2.5% (v/v) TIS and 2.5% (v/v) water for 1 h atRT. Compound of formula (ex-5h) was obtained, the structure wasconfirmed by RP-HLPC analysis method B3

Phe-Ala-Leu-NH₂  (ex-5h)

i) Coupling of Compound of Formula (Ex-4) with Compound of Formula(Ex-5G4) by HSPPS

A mixture of compound of formula (ex-4) (84 mg, 1 eq), preparedaccording to example 4, HOBt (53 mg, 3.2 eq) and DIPCDI (53 microliter,3.1 eq) in DCM (2 ml) was stirred for 15 min at RT, then added to asolution of compound of formula (ex-5g4), prepared according to example5 g4), (100 mg, 1.1 eq) in DCM (1 ml), and then the mixture was stirredat RT for 4 h. The coupling was monitored by HPLC method B3.

The reaction mixture was washed with aqueous saturated NaHCO₃ (2×40 ml),1M KHSO₄aqueous solution (2×40 ml) and aqueous saturated NaCl (2×40 ml).

The organic phase was dried over MgSO₄ and concentrated under reducedpressure to obtain 328.5 mg of compound of formula (ex-5i) as an oil in55% purity, as determined by analytical RP-HPLC (method description B4),consisting of 2 peaks of 26.8% and 28.2% each due to the 2diastereoisomers caused by the chirality of the Ramage handle group.

(The core peptide below is disclosed as SEQ ID NO: 5.)

Example 6 Attachment of the Diketopiperazine Group Forming DipeptidylLinker to the HMPS Resin. Preparation of Compounds of Formulae(Ex-6e3-D1), (Ex-6e3-d2), (Ex-6F1) and (ex-6f1) a) Introduction of theSecond Amino Acid L-Lys

A mixture of Trt-Lys(Fmoc)-OH (10.08 g, 2 eq), HOBt (3.79 g, 3 eq) andDIPCDI (3.12 g, 3 eq) in DMF (100 ml) was stirred for 5 min at RT, thenadded to a resin prepared according to example 5 b2). The mixture wasstirred at RT for 17 h.

Then, the resin was washed with DMF (5×1 min; 150 ml each), and the Fmocgroup was removed according to method Fmoc-gr-rem.

b) Introduction of Handle Group, Spacer Group and Amino Acids by SPPS

The handle group, spacer group and each amino acid was reacted in areaction cycle. The reaction steps in one reaction cycle for theincorporation of one amino acid follow the reaction cycle description(vii).

c) Reaction Cycle Description (VII) for Elongation Sequences d1) and d2)

Each time, a mixture of the spacer group providing Fmoc-TTDS-OH in caseof d1), of the handle group providing Fmoc-Rink-OH or of the respectiveFmoc-Xaa-OH, according to the elongation sequence, together with HOBt(3.79 g, 3 eq) and DIPCDI (3.12 g, 3 eq) in DMF (100 ml) was stirred for5 min at RT and then added firstly to a resin prepared according toexample 6a) and then to the resin prepared in the preceding step in theelongation sequence.

The mixture was stirred at RT for 1 to 4 h. Then, the resin was washedwith DMF (5×1 min; 150 ml each. 6× instead of 5× when Fmoc-Xaa-OH wasFmoc-Ala-OH) and the Fmoc group was removed according to methodFmoc-gr-rem.

Only before Fmoc group removal of the last Fmoc-Xaa-OH of the respectiveelongation sequence d1) or d2), a small portion of obtainedpeptidyl-resin (5 mg) was cleaved from the Rink amide handle group bytreating the resin with 1 ml of a mixture consisting of 95% (v/v) TFA,2.5% (v/v) TIS and 2.5% (v/v) H₂O for 1 h at RT. Compound of formula(ex-6c-d1) was obtained in 46.7% purity by elongation sequence d1), andcompound of formula (ex-6c-d2) in 65.4% purity by elongation sequenced2) (RP-HPLC, method description B3).

Fmoc-Phe-Ala-Leu-NH₂  (ex-6c-d1)

Fmoc-Tyr(tBu)-His(Trt)-Leu-NH₂  (ex-6c-d2)

d) Incorporation of Amino Acids by SPPS

The sequence of incorporation of the amino acids was:

Elongation sequence d1)

Fmoc-TTDS-OH/Fmoc-Rink-OH/ Fmoc-Xaa-OH mixture stirred for 1.Fmoc-TTDS-OH (5 g) 4 h 2. Fmoc-Rink-OH (9.11 g) 1 h 3. Fmoc-Leu-OH (5.83g) 1 h 4. Fmoc-Ala-OH (5.43 g) 1 h 5. Fmoc-Phe-OH (3.69 g)Elongation Sequence d2)

Fmoc-Xaa-OH/Fmoc-Rink-OH mixture stirred for 1. Fmoc-Rink-OH (9.10 g) 1h 2. Fmoc-Leu-OH (5.83 g) 2 h 3. Fmoc-His(Trt)-OH (10.23 g) 1 h 4.Fmoc-Tyr(tBu)—OH (7.59 g) 1 hE) Trt Protecting Group Removal of the L-Lys of the DiketopiperazineGroup Forming Dipeptidyl Linker, Formation of the DiketopiperazineResidue Comprising C-Terminal Protecting Group and Cleavage from theResin

Formation of the diketopiperazine residue comprising C-terminalprotecting group and cleavage of the DKP-peptides from the resinsprepared according to examples 6 c) in combination with examples 6 d1)and 6 d2) respectively, are brought about in analogous manner asdescribed in example 1.4a with the amounts of reagents and solventadapted to the amount of peptidyl-resin:

e1) Trt Group Deprotection

Treatment of the compound prepared according to example 6 c) incombination with examples 6 d1) and 6 d2) respectively with 0.2% (v/v)TFA in DCM (2×5 min, 100 ml each).

e2) Neutralization

Then treatment with 5% (v/v) DIEA in DCM (2×5 min; 100 ml each), DCM(2×1 min; 100 ml each) and THF (2×1 min; 100 ml each).

E3) Formation of the Diketopiperazine Residue Comprising C-TerminalProtecting Group and Cleavage from the Resin

Treatment according to the procedure of example 5 g3). Oils wereobtained, 0.83 g of compound of formula (ex-6e3-d1) in case of thestarting material from example 6 d1), and 2.03 g of compound of formula(ex-6e3-d2) in case of the starting material from example 6 d2).

F1) Coupling of Compound of Formula (Ex-4) with Compound of Formula(Ex-6E3-Dl) by HSPPS

A mixture of compound of formula (ex-4) (255 mg, 1 eq), preparedaccording to example 4, HOBt (160 mg, 3 eq) and DIPCDI (161 microliter,3 eq) in DCM (5 ml) was stirred for 60 min at RT, then added to asolution of compound of formula (ex-6e3-d1) (401 mg, 1.0 eq), preparedaccording to example 6e3), in DCM (1 ml), and then stirred at RT for 2h. The coupling was monitored by HPLC method B3. The reaction mixturewas washed with aqueous saturated NaHCO₃ (2×40 ml), 1M KHSO₄ aqueoussolution (2×40 ml) and aqueous saturated NaCl (2×40 ml). The organicphase was dried over MgSO₄ and concentrated under reduced pressure toobtain 368.5 mg of compound of formula (ex-6f1) as an oil.

(The core peptide below is disclosed as SEQ ID NO: 7.)

f2) Addition of Compound of Formula (ex-4) with Compound of Formula(ex-6e3-d2) by HSPPS

A mixture of compound of formula (ex-4) (295 mg, 1 eq), preparedaccording to example 4, HOBt (190 mg, 3 eq) and DIPCDI (188 microliter,3 eq) in DCM (5 ml) was stirred for 20 min at RT, then added to asolution of compound of formula (ex-6e3-d2) (502 mg, 1.0 eq), preparedaccording to example 6e3), in DCM (1 ml), and then stirred at RT for 3.5h. The coupling was monitored by HPLC method B3.

The reaction mixture was washed with aqueous saturated NaHCO₃ (2×40 ml),1M KHSO₄aqueous solution (2×40 ml) and aqueous saturated NaCl (2×40 ml).The organic phase was dried over MgSO₄ and concentrated under reducedpressure to obtain 372.8 mg of compound of formula (ex-6f2) as an oil.(The core peptide below is disclosed as SEQ ID NO: 6.)

Example 7 Use of the Handle Group HMPA. Preparation of Compound ofFormula (Ex-7h3) a) Pre-Treatment of the HMPS Resin

HMPS resin (103.6 mg) was swelled with DCM (5×1 min; 3 ml each) and DMF(5×1 min; 3 ml each) at RT and then filtered.

b) Introduction of the First Amino Acid (D-Pro) of the DiketopiperazineGroup Forming Dipeptidyl Linker on the Resin

Fmoc-D-Pro-OH (132 mg, 4 eq) and DIPCDI (30 microliter, 2 eq) in DCM/DMF(15:1 (v/v), 2.5 ml) was added to a resin prepared according to example7a). Then, DMAP (4.8 mg, 0.4 eq) in DCM (0.5 ml) was added and left tostand at RT for 2 h. The first amino acid was re-coupled usingFmoc-D-Pro-OH (132 mg, 4 eq) and DIPCDI (30 microliter, 2 eq) in DCM/DMF(15:1 (v/v), 2.5 ml) for 16 h at RT. After coupling, the resin waswashed with DCM (5×1 min; 3 ml each) and with DMF (5×1 min; 3 ml each).Then, the resin was capped using acetic anhydride (46 microliter, 5 eq)and DIEA (86 microliter, 5 eq) in DMF (2.5 ml) for 30 min at RT. Aftercapping, the resin was washed with DCM (5×1 min; 3 ml each) and with DMF(5×1 min; 3 ml each). Then the Fmoc group was removed by the methodFmoc-gr-rem.

A 0.98 mmol/g resin loading was determined by UV quantification (methoddescription A; V_(A): 100 ml, V_(B): 10 ml and V_(C): 1.4 ml).

c) Introduction of the Second Amino Acid (L-Dpr)

A mixture of Trt-L-Dpr(Fmoc)-OH (173 mg, 3 eq), HOBt (47 mg, 3 eq) andDIPCDI (47 microliter, 3 eq) in DMF (2 ml) was shaken for 5 min at RT,then added to a resin prepared according to example 7b). The mixture wasleft to stand at RT for 1 h. No re-coupling was required according tothe ninhydrin test (method C). Then the resin was washed with DMF (5×1min; 3 ml each) and with DCM (5×1 min; 3 ml each). Then the Fmoc groupwas removed by the method Fmoc-gr-rem.

d) Introduction of HMPA Handle Group

A mixture of HMPA (55 mg, 3 eq), HOBt (47 mg, 3 eq) and DIPCDI (47microliter, 3 eq) in DMF (2 ml) was added to a resin prepared accordingto example 7c), and then left to stand at RT for 1 h. No re-coupling wasrequired, according to the ninhydrin test (method C). Then the resin waswashed with DMF (5×1 min; 3 ml each) and with DCM (5×1 min; 3 ml each).

e) Introduction of Fmoc-Xaa-OH by SPPS

Starting with a resin prepared according to example 7d), 1. Fmoc-Leu-OHwas incorporated following the reaction cycle description (viii), andthen 2. Fmoc-Ala-OH and 3. Fmoc-Phe-OH were respectively incorporatedfollowing the reaction description (ix).

f1) Reaction cycle description (viii)

Fmoc-Leu-OH (144 mg, 4 eq) and DIPCDI (30 microliter, 2 eq) in DCM/DMF(15:1 (v/v), 2.5 ml) was added to a resin prepared according to example7d). Then, DMAP (4.8 mg, 0.4 eq) in DCM (0.5 ml) was added and left tostand at RT for 2 h. The amino acid was re-coupled using Fmoc-Leu-OH(144 mg, 4 eq) and DIPCDI (30 microliter, 2 eq) in DCM/DMF (15:1 (v/v),2.5 ml) for 16 h at RT. After coupling, the resin was washed with DCM(5×1 min; 3 ml each) and with DMF (5×1 min; 3 ml each). Then, the resinwas capped using acetic anhydride (46 microliter, 5 eq) and DIEA (86microliter, 5 eq) in DMF (2.5 ml) for 30 min at RT. After capping, theresin was washed with DCM (5×1 min; 3 ml each) and with DMF (5×1 min; 3ml each). Then the Fmoc group was removed by the method Fmoc-gr-rem. A0.94 mmol/g resin loading was determined by UV quantification (methoddescription A; V_(A): 100 ml, V_(B): 10 ml and V_(C): 1.4 ml).

f2) Reaction Cycle Description (IX)

A mixture of the respective Fmoc-Xaa-OH (3 eq), HOBt (47 mg, 3 eq) andDIPCDI (47 microliter, 3 eq) in DMF (2 ml) was added to a resin and thenleft to stand at RT for 1 h. No re-coupling was required, according tothe ninhydrin test (method C). Then the resin was washed with DMF (5×1min; 3 ml each) and with DCM(5×1 min; 3 ml each). The Fmoc group wasremoved by according to method Fmoc-gr-rem.

g) Analysis—Cleavage of the Peptide from the Handle Group and Therebyfrom the Resin

A small portion of resin (5 mg), obtained according to example 7e), wascleaved from the resin by treating the resin with 1 ml of a mixture of95% (v/v) TFA, 2.5% (v/v) TIS and 2.5% (v/v) water for 1 h at RT. TheRP-HLPC analysis confirmed identity of Phe-Ala-Leu-NH₂(methoddescription B1, 5 to 100 of mobile phase B).

h) Trt Protecting Group Removal of the L-Dpr of the DiketopiperazineGroup Forming Dipeptidyl Linker, Formation of the DiketopiperazineResidue Comprising C-Terminal Protecting Group and Cleavage from theResin

Formation of the diketopiperazine residue comprising C-terminalprotecting group and cleavage of the DKP-peptides from the resinprepared according to example 7e), is brought about in analogous manneras described in example 1.4a with the amounts of reagents and solventadapted to the amount of peptidyl-resin:

h1) Trt Group Deprotection

Treatment of the compound prepared according to example 7 e) with 0.2%(v/v) TFA, 2% (v/v) TIS in DCM (2×5 min, 2 ml each).

h2) Neutralization

The treatment with 5% (v/v) DIEA in DCM (2×5 min; 2 ml each)

h3) Formation of the Diketopiperazine Residue Comprising C-TerminalProtecting Group and Cleavage from the Resin

Then treatment with 5% (v/v) piperidine in THF (2×5 min; 2 ml each).

THF was removed by evaporation under vacuum and the resulting compoundof formula (ex-7h3) was analyzed by RP-HPLC-ESMS (method description B2,5 to 100 of mobile phase B, [(M+H)/2]+: 679, where M is the MW ofcompound of formula (ex-7h3)).

Sequence Listing Free Text

<210> 1<223> SEQ ID 1 is abbreviated with [T20-1-36]<210> 2<223> SEQ ID 2 is abbreviated with [T20-27-36]<210> 3<223> SEQ ID 3 is abbreviated with [T20-17-26]<210> 4<223> SEQ ID 4 is abbreviated with [T20-17-36]<210> 5<223> SEQ ID 5 is comprised in formulae (ex-5i)<210> 6<223> SEQ ID 6 is comprised in formula (ex-6f2)<210> 7<223> SEQ ID 6 is comprised in formula (ex-6f1)

1-13. (canceled)
 14. A method for the preparation of a DKP-PG forminglinker DKP-L, wherein the DKP-PG forming linker DKP-L comprises a handlegroup HG, an optional spacer group SG, and a dipeptide DPR, whereinDKP-PG is a protecting group comprising the handle group HG, theoptional spacer group SG, and a diketopiperazine residue DKP; whereinDKP is derived from the dipeptide DPR; wherein SG is a spacer groupconventionally used in peptide chemistry; wherein DPR consists of aC-terminal residue Xaa1 and an N-terminal residue Xaa2, wherein Xaa2 hasa side chain functional group FG; wherein Xaa1 is selected from thegroup consisting of naturally occurring alpha amino acid residues,alpha-N-methylamino acid residues, L-Hpr residue, D-Hpr residue, DL-Hprresidue, 2-(C₁₋₅-alkyl)-D-amino acid residues, 2-(C₁₋₅-alkyl)-L-aminoacid residues, 2-(C₁₋₅-alkyl)-DL-amino acid residue and an HypX residue;wherein HypX is a compound of formula:

wherein X is O, S or C(R13)R14; R5, R7, R12, R13 and R14 are identicalor different and independently from each other selected from the groupconsisting of hydrogen, C₁₋₄ alkyl and O—R8; R8 is a protecting groupconventionally used for side chain protection in peptide chemistry, or asubstituent of formula (Sub-R8);

wherein m8 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R9 is C₁₋₄ alkyl; whereinXaa2 is selected from the group consisting of L-Lys residue, D-Lysresidue, DL-Lys residue, L-Orn residue, D-Orn residue, DL-Orn residue,L-4-aminoproline residue, D-4-aminoproline residue, DL-4-aminoprolineresidue, L-alpha,gamma-diaminobutanoic acid residue,D-alpha,gamma-diaminobutanoic acid residue, DL-alpha,gamma-diamino

butanoic acid residue, L-alpha,beta-diaminopropanoic acid residue,D-alpha,beta-diaminopropanoic acid residue,DL-alpha,beta-diaminopropanoic acid residue, L-Ser residue, D-Serresidue, DL-Ser residue, L-Thr residue, D-Thr residue, DL-Thr residue,L-Cys residue, D-Cys residue, DL-Cys residue, L-homocysteine residue,D-homocysteine residue, DL-homocysteine residue, L-Asp residue, D-Aspresidue, DL-Asp residue, L-Glu residue, D-Glu residue and DL-Gluresidue; wherein HG is either directly connected to a side chainfunctional group of Xaa2 (FG), or, if a SG is present, HG is connectedto SG and SG is connected to FG; wherein HG is a handle group selectedfrom the group consisting of HGF-I, HGF-II, HGF-III, HGF-IV, HGF-V andHGF-VI,

wherein (*) denotes the bond between HG and the carbonyl carbon or theside chain of the C-terminal amino acid of PEP-C, (**) denotes the bondbetween HG and SG when a SG is present, or denotes the bond between HGand FG when no SG is present; R1, R2, R3, R4, R10 and R11 are identicalor different and independently from each other selected from the groupconsisting of hydrogen and O—C₁₋₄ alkyl, s1-1, s2, s3, s4 and s6 areidentical or different and independently from each other selected fromthe group consisting of 1, 2, 3 and 4, s5-1 is 0, 1, 2, 3 or 4, s1-2,s5-2 and s5-3 are identical or different and independently from eachother 0 or 1, T1-1 is O or NH, T1-2 and T5-1 are O; wherein said methodcomprises coupling Xaa2 to Xaa1; optionally coupling SG to Xaa2, if SGis present; coupling HG either to SG, if SG is present, or to Xaa2. 15.The method according to claim 14, wherein HG is selected from the groupconsisting of HGF-I, HGF-IV and HGF-VI.
 16. The method according toclaim 14, wherein HG is selected from the group consisting of HG-Ia,HG-Ib, HG-Ic, HG-Id, HG-II, HG-III, HG-IVa, HG-IVb, HG-Va, HG-Vb andHG-VI,

wherein (*) and (**) are as defined in claim
 14. 17. The methodaccording to claim 14, wherein SG is a spacer group selected from thegroup consisting of SG-I, SG-II, SG-III, SG-IV SG-V, SG-VI, and SG-VII;

wherein m1, m5, m6, m7, m9, m10, mil and m12 are identical or differentand independently from each other an integer of 1 to 500; m2, m3 and m4are identical or different and independently from each other 1, 2, 3 or4, (***) is the bond from SG to HG when a SG is present, (****) is thebond between SG and Xaa2 when a SG is present, wherein HG and Xaa2 aredefined according to claim
 14. 18. The method according to claim 14,wherein Xaa1 is selected from the group consisting of L-N-methylglycineresidue, D-N-methylglycine residue, DL-N-methylglycine residue,L-N-methylphenylalanine residue, D-N-methylphenylalanine residue,DL-N-methylphenylalanine residue, L-Pro residue, D-Pro residue, DL-Proresidue, side chain protected L-4Hyp residue, side chain protectedD-4Hyp residue, side chain protected DL-4Hyp residue, L-4Hpr residue,D-Hpr residue and DL-Hpr residue.
 19. A method according to claim 14,wherein Xaa2 is selected from the group consisting of L-Lys residue,D-Lys residue, DL-Lys residue, L-Orn residue, D-Orn residue, DL-Ornresidue, L-4-aminoproline residue, D-4-aminoproline residue,DL-4-aminoproline residue, L-alpha,gamma-diaminobutanoic acid residue,D-alpha,gamma-diaminobutanoic acid residue,DL-alpha,gamma-diaminobutanoic acid residue,L-alpha,beta-diaminopropanoic acid residue,D-alpha,beta-diaminopropanoic acid residue,DL-alpha,beta-diaminopropanoic acid residue, L-Ser residue, D-Serresidue, DL-Ser residue, L-Thr residue, D-Thr residue, DL-Thr residue,L-Cys residue, D-Cys residue, DL-Cys residue, L-homocysteine residue,D-homocysteine residue, DL-homocysteine residue, L-Asp residue, D-Aspresidue, DL-Asp residue, L-Glu residue, D-Glu residue and DL-Gluresidue.
 20. A compound DKP-L as defined according to claim
 14. 21. Amethod for the preparation of a DKP-L-ResinA, wherein DKP-L-ResinAcomprises a ResinA connected to a DKP-PG forming linker DKP-L, whereinResinA is a resin used conventionally as solid phase in SPPS, whereinthe DKP-PG forming linker DKP-L comprises a handle group HG, an optionalspacer group SG, and a dipeptide DPR; wherein DKP-PG is a protectinggroup comprising the handle group HG, the optional spacer group SG, anda diketopiperazine residue DKP; wherein DKP is derived from thedipeptide DPR; wherein DPR consists of a C-terminal residue Xaa1 and anN-terminal residue Xaa2, wherein Xaa2 has a side chain functional groupFG; wherein the carboxylic acid group of Xaa1 is connected to ResinA;wherein SG is a spacer group conventionally used in peptide chemistry;wherein Xaa1 is selected from the group consisting of naturallyoccurring alpha amino acid residues, alpha-N-methylamino acid residues,L-Hpr residue, D-Hpr residue, DL-Hpr residue, 2-(C₁₋₅-alkyl)-D-aminoacid residues, 2-(C₁₋₅-alkyl)-L-amino acid residues,2-(C₁₋₅-alkyl)-DL-amino acid residue and an HypX residue; wherein HypXis a compound of formula:

wherein X is O, S or C(R13)R14; R5, R7, R12, R13 and R14 are identicalor different and independently from each other selected from the groupconsisting of hydrogen, C₁₋₄ alkyl and O—R8; R8 is a protecting groupconventionally used for side chain protection in peptide chemistry, or asubstituent of formula (Sub-R8);

wherein m8 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R9 is C₁₋₄ alkyl; whereinXaa2 is selected from the group consisting of L-Lys residue, D-Lysresidue, DL-Lys residue, L-Orn residue, D-Orn residue, DL-Orn residue,L-4-aminoproline residue, D-4-aminoproline residue, DL-4-aminoprolineresidue, L-alpha,gamma-diaminobutanoic acid residue,D-alpha,gamma-diaminobutanoic acid residue, DL-alpha,gamma-diamino

butanoic acid residue, L-alpha,beta-diaminopropanoic acid residue,D-alpha,beta-diaminopropanoic acid residue,DL-alpha,beta-diaminopropanoic acid residue, L-Ser residue, D-Serresidue, DL-Ser residue, L-Thr residue, D-Thr residue, DL-Thr residue,L-Cys residue, D-Cys residue, DL-Cys residue, L-homocysteine residue,D-homocysteine residue, DL-homocysteine residue, L-Asp residue, D-Aspresidue, DL-Asp residue, L-Glu residue, D-Glu residue and DL-Gluresidue; wherein HG is either directly connected to a side chainfunctional group of Xaa2 (FG), or, if a SG is present, HG is connectedto SG and SG is connected to FG; wherein HG is a handle group selectedfrom the group consisting of HGF-I, HGF-II, HGF-III, HGF-IV, HGF-V andHGF-VI,

wherein (*) denotes the bond between HG and the carbonyl carbon or theside chain of the C-terminal amino acid of PEP-C, (**) denotes the bondbetween HG and SG when a SG is present, or denotes the bond between HGand FG when no SG is present; R1, R2, R3, R4, R10 and R11 are identicalor different and independently from each other selected from the groupconsisting of hydrogen and O—C₁₋₄ alkyl, s1-1, s2, s3, s4 and s6 areidentical or different and independently from each other selected fromthe group consisting of 1, 2, 3 and 4, s5-1 is 0, 1, 2, 3 or 4, s1-2,s5-2 and s5-3 are identical or different and independently from eachother 0 or 1, T1-1 is O or NH, T1-2 and T5-1 are O; wherein said methodcomprises method (X1) or method(X2); wherein method(X1) comprisescoupling the amino acid Xaa1 to ResinA; coupling the amino acid Xaa2 toXaa1; optionally coupling SG to the side chain of Xaa2, if SG is presentin DKP-L-ResinA; and coupling HG either to SG, if SG is present inDKP-L-ResinA, or to Xaa2; and wherein method(X2) comprises couplingDKP-PG forming linker DKP-L to ResinA.
 22. The method according to claim21, wherein HG is selected from the group consisting of HGF-I, HGF-IVand HGF-VI.
 23. The method according to claim 21, wherein HG is selectedfrom the group consisting of HG-Ia, HG-Ib, HG-Ic, HG-Id, HG-II, HG-III,HG-IVa, HG-IVb, HG-Va, HG-Vb and HG-VI,

wherein (*) and (**) are as defined in claim
 21. 24. The methodaccording to claim 21, wherein SG is a spacer group selected from thegroup consisting of SG-I, SG-II, SG-III, SG-IV SG-V, SG-VI, and SG-VII;

wherein m1, m5, m6, m7, m9, m10, m11 and m12 are identical or differentand independently from each other an integer of 1 to 500; m2, m3 and m4are identical or different and independently from each other 1, 2, 3 or4, (***) is the bond from SG to HG when a SG is present, (****) is thebond between SG and Xaa2 when a SG is present, wherein HG and Xaa2 aredefined according to claim
 21. 25. The method according to claim 21,wherein Xaa1 is selected from the group consisting of L-N-methylglycineresidue, D-N-methylglycine residue, DL-N-methylglycine residue,L-N-methylphenylalanine residue, D-N-methylphenylalanine residue,DL-N-methylphenylalanine residue, L-Pro residue, D-Pro residue, DL-Proresidue, side chain protected L-4Hyp residue, side chain protectedD-4Hyp residue, side chain protected DL-4Hyp residue, L-4Hpr residue,D-Hpr residue and DL-Hpr residue.
 26. A method according to claim 21,wherein Xaa2 is selected from the group consisting of L-Lys residue,D-Lys residue, DL-Lys residue, L-Orn residue, D-Orn residue, DL-Ornresidue, L-4-aminoproline residue, D-4-aminoproline residue,DL-4-aminoproline residue, L-alpha,gamma-diaminobutanoic acid residue,D-alpha,gamma-diaminobutanoic acid residue,DL-alpha,gamma-diaminobutanoic acid residue,L-alpha,beta-diaminopropanoic acid residue,D-alpha,beta-diaminopropanoic acid residue,DL-alpha,beta-diaminopropanoic acid residue, L-Ser residue, D-Serresidue, DL-Ser residue, L-Thr residue, D-Thr residue, DL-Thr residue,L-Cys residue, D-Cys residue, DL-Cys residue, L-homocysteine residue,D-homocysteine residue, DL-homocysteine residue, L-Asp residue, D-Aspresidue, DL-Asp residue, L-Glu residue, D-Glu residue and DL-Gluresidue.
 27. The method according to claim 21, wherein ResinA isselected from the group consisting of hydroxymethylpolystyrene (HMPS)resins, polyethylene glycol (PEG) based resins, polystyrene resin,p-benzyloxybenzyl alcohol resins, chloromethylpolystyrene-divinylbenzene resins, and poly(vinylalcohol)-graft-poly(ethylene glycol) (PVA-g-PEG) resins.
 28. A compoundDKP-L-ResinA as defined according to claim 21.